TESE DE DOUTORAMENTO Recuperación De Suelos De Mina

2017

Recuperación de suelos de mina combinando la aplicación de suelos combinando de mina la aplicación Recuperación TESE DE DOUTORAMENTO ” Recuperación de suelos de mina combinando la aplicación de enmiendas elaboradas con residuos con la fitoremediación Rubén Forján Castro 2017

“TESE DE DOUTORAMENTO DE “TESE

Rubén Forján CastroRubén elaboradas la con fitoremediación residuos enmiendas con de

Escola Internacional de Doutoramento

Rubén Forján Castro

TESE DE DOUTORAMENTO

Recuperación de suelos de mina combinando la aplicación de enmiendas elaboradas con residuos con la fitoremediación

Dirixida pola doutora:

Emma Fernández Covelo

2017

Rubén Forján Castro foi beneficiario dunha beca predouctoral da Universidade de Vigo, entre os anos 2014 e 2016. (Aplicación orzamentaria 00VI 131H 641.03). E actualmente contratado a cargo proxecto 10404 CANICOUVA. Este traballo tamén foi financiado polo proxecto CGL2016-78660-R

A mi familia

Índice

1. Introducción……………………………………………………………….27

1.1 Degradación y contaminación (estado)……………………………………..…27

1.2 Minería de superficie………………………………………………………….29

1.2.1 La mina de Touro……………………………………………………..31

1.2.2 Residuos mineros…………………………………………………...…32

1.3 Producción y gestión de residuos………………………………...... …33

1.4 Residuos y Enmiendas…..………………………………………………….….36

1.4.1 Compost……………………………………………………...………..37

1.4.2 Tecnosoles………………………………………………………...…..39

1.4.3 Biochar…………………………………………………….………….43

1.4.4 Capacidad de sorción de las enmiendas elaboradas con residuos

(compost, tecnosol, biochar)…………………………………….…….46

1.5 Fitoremediación………………………………………………………………..48

1.5.1 Fitoestabilización………………………………………...………………48

2. Estudios previos

2.1 Effect of amendments made of waste materials in the physical and chemical recovery of mine soil…………………………….……………………………………………..55

Abstract…………………………………………………………………………………56

1. Introduction………………………………………………………………………...55

2. Material and methods………………………………………………………………56

3. Results……………………………………………………………………………...57

3.1 Physico-chemical characteristics of the mine soil (S), amendments (A1 and A2)

and treated soils (SA1 and SA2) at the initial time……………………………57

3.2 Evolution of the physical–chemical characteristics in the amended and

unamended soil over the 3-month period……………………………………...58

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4. Discussion………………………………………………………………………….60

5. Conclusions………………………………………………………………………...60

6. References………………………………………………………………………….60

2.2 Contribution of waste and biochar amendment to the sorption of metals in a copper mine tailing………………………………………………………………………….....65

Abstract………………………………………………………………...……………….65

1. Introduction…………………………………………………………………...……65

2. Material and methods………………………………………………………...…….66

2.1 Study area and amendments………………………………...………...……….66

2.2 Experiment design and soil chemical analyses…………………..…………….66

2.3 Sorption experiment and construction of isotherms………………………...…66

2.4 Statistical analyses…………………………………………………………..…66

3. Results……………………………………………………………………………...67

3.1 Characteristics of the mine tailing (S), amendment (T) and biochar (B)……...67

3.2 Characteristics of the mine tailing (S), mine tailing amended with amendment

mixtures (STB20, STB40%, STB60%) and biochar amendment (TB100%)…67

3.3 Sorption isotherms of Cu, Pb and Zn……………………………………….....67

3.4 Estimation of the sorption capacity using the distribution coefficient Kr…..…68

3.5 Selectivity sequences for Cu, Pb and Zn………………………………………68

4. Discussion……………………………………………………………………….…68

4.1 Characteristics of the mine tailing (S), mine spoil material amended with

amendment mixtures (STB20, STB40%, STB60%) and biochar amendment

(TB100%)……………………………………………………………………...68

4.2 Sorption capacity of mine tailing for Ni, Pb and Zn…………...... …………....69

4.3 Selectivity sequences for Cu, Pb and Zn….70

5. Conclusions………………………………………………………………………….69

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6. References………………………………………………………………………..….70

2.3 Contributions of a compost-biochar mixture to the metal sorption capacity of a mine tailing………………………………………….………………………………………….75

Abstract…………………………………………………………………………………75

1. Introduction………………………………………………………………………...75

2. Material and methods……………………………………………………………....76

2.1 Study area and experiment design………………………………………….….76

2.2 Chemical analyses……………………………………………………………..76

2.3 Sorption experiment and construction of isotherms…………………………...76

2.4 Statistical analyses………………………………………………………….….77

3. Results…………………………………………………………………………..….77

3.1 Characteristics of the mine tailing (S), compost (C) and biochar (B)………....77

3.2 Characteristics of the mine tailing (S), amended mine tailing (SCB20 %, SCB40

%, SCB60 %) and the compost + biochar positive control (CB100 %)……….78

3.3 Sorption capacity of Cu, Pb and Zn by the amended and unamended mine

tailing……………………………………………………….………………….78

4. Discussion………………………………………………………………………….79

4.1 General characteristics of the studied samples……………………………...…79

4.2 Sorption capacity of the studied samples for Cu, Pb and Zn……………….….80

5. Conclusions………………………………………………………………………...81

6. References………………………………………………………………………….81

3. Justificación y objetivos…………………………………………...………87

4. Capítulo 1 Changes in phytoavailable concentrations in a mine soil following the application of technosols and biochar with Brassica juncea L...... 93

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Abstract…………………………………………………………………………………93

1. Introduction………………………………………………………………………...94

2. Material and Methods……………………………………………………………...96

2.1 Soil sampling…………………………………………………………………..96

2.2 Greenhouse Experiment……………………………………………………….98

2.3 Soil Analysis………………………………………………………………..…99

2.4 Plant Growth and Determination of Metals in Plant Tissues……..….………100

2.5 Statistical Analysis……………………………………………………...……101

3. Results…………………………………………………………………………….101

3.1 General Characteristics of the Settling Pond Soil (S), Sand (SS), Technosol (T),

and Biochar (B)…………………………………………………………...….101

3.2 Evolution of the Pseudototal Concentrations of Cu, Pb, Ni, Zn at Three Heights

and over the 11-Month Period………………………………………………..103

3.3 Evolution of CaCl2-Extractable (Phytoavailable) Contents of Cu, Pb, Ni, Zn at

Three Heights and over the 11-Month Period………………………………..105

3.4 Harvestable Amounts of Cu, Pb, Ni, Zn and Determination of Metals in Plant

Tissues: Translocation Factor (TF) and Transfer Coefficient (TC)………….108

3.4.1 Translocation Factor (TF)…………………………………………....108

3.4.2 Transfer Coefficient (TrC)…………………………………………..112

4. Discusion……………………………………………………………………...…..113

4.1 Evolution of the Pseudototal Contents of Cu, Pb, Ni, Zn at Three Heights and

Over the 11-Month Period……………………………………………………113

4.2 Evolution of the CaCl2-Extractable (Phytoavailable) Concentrations of Cu, Pb,

Ni, Zn at Three Heights and over the 11-Month Period…………..………….114

4.3 Uptake and Transfer of Metals to Mustards………………………………….116

4.3.1 Harvestable Amounts of Cu, Pb, Ni, Zn…………………………..…116

4.3.2 Transfer Coefficient (TrC)……………………………………….….116

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4.3.3 Translocation Factor (TF)……………………………………………117

5. Conclusion………………………………………………………………………..117

6. References………………………………………………………………………...118

5. Capítulo 2 Application of Compost and Biochar with Brassica juncea L. to

Reduce Phytoavailable Concentrations in a Settling Pond Mine Soil…………..………...127

Abstract………………………………………………………………………………..127

1. Introduction……………………………………………………………………….127

2. Materials and Methods………………………………………………………...….128

2.1 Soil Sampling…………………………………………………………...……128

2.2 Greenhouse Experiment………………………………………………...……129

2.3 Soil Analysis…………………………………………………………….……130

2.4 Plant Growth and Determination of Metals in Plant Tissues………………...130

2.5 Statistical Analysis………………………………………………………..….130

3. Results…………………………………………………………………………….131

3.1 General Characteristics of Settling Pond Soil (S), Sand (SS), Compost (C), and

Biochar (B)……………………………………………………………..…….131

3.2 Evolution of the Pseudo‑total Contents of Cu, Pb, Ni, and Zn at the Three

Different Depths and Over the 11‑Month Period……………………...……..131

3.3 Evolution of the CaCl2‑Extractable (Phytoavailable) Contents of Cu, Pb, Ni,

and Zn at the Three Different Depths and over the 11‑Month Period………..132

3.4 Harvestable Amounts of Cu, Pb, Ni, and Zn and Determination of Metals in

Plant Tissues: Translocation Factor (TF) and Transfer Coefficient (TC)……134

3.4.1 Harvestable Amounts of Cu, Pb, Ni, and Zn in Brassica juncea L..134

3.4.2 Transfer Coefficient (TC)…………………………………………....134

3.4.3 Translocation Factor (TF)………………..…………………………..137

4. Discussion………………………………………………………………………...137

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4.1 Changes in Pseudo‑Total Soil Cu, Pb, Ni, and Zn at the Three Different Depths

and Over the 11‑Month Period……………………………………………….137

4.2 Changes in CaCl2‑Extractable (Phytoavailable) Concentrations of Cu, Pb, Ni,

and Zn at the Three Different Depths and Over the 11‑Month Period…….....137

4.3 Harvestable Amounts of Cu, Pb, Ni, and Zn and Metal Concentrations in Plant

Tissues: Translocation Factor (TF) and Transfer Coefficient (TC)……….....138

5. Conclusions……………………………………………………………………….138

6. References………………………………………………………………………...138

6. Anexos……………………………………………………………...………………143

6.1 Anexo I Increasing the nutrient content in a mine soil through the application of technosol and biochar and grown with Brassica juncea L.………………..……………147

Abstract………………………………………………………………………………..147

1. Introduction……………………………………………………………………….148

2. Material and Methods…………………………………………………………….151

2.1 Soil sampling and amendments……………...……………………………….151

2.2 Greenhouse experiment………………………………………………………152

2.3 Soil, technosol and biochar analysis………………………………………….154

2.4 Harvested biomass and height of Brassica juncea L………………………....154

2.5 Statistical analysis…………………………………………………………....155

3. Results………………………………………………………………………….....155

3.1 General characteristics of the settling pond soil (S), sand (SS), technosol (T),

and biochar (B)……………………………………………………………….155

3.2 Evolution of the pH at the three depths and over the 11-month period………157

3.3 Evolution of Total Carbon (TC) at three depths and over the 11-month

period…………………………………………………………………………157

3.4 Evolution of Total Nitrogen (TN) at three depths and over the 11-month

period…………………………………………………………………………158

Índice

3.5 Evolution of the cation exchange capacity (CEC), base saturation (%), and

aluminium saturation (%) at three depths and over the 11-month period……159

3.6 Evolution of nutrients in three depths and over the 11-month period………..160

3.7 Limiting factors for plant production in depth 0-15cm………………………163

3.8 Harvested biomass of Brassica juncea L………………………………….…164

4. Discussion……………………………………………………………………..….165

4.1 Evolution of the pH at the three depths and over the 11-month period…..…..165

4.2 Evolution of Total Carbon (TC) at three depths and over the 11-month

period…………………………………………………………………………166

4.3 Evolution of Total Nitrogen (TN) at three depths and over the 11-month

period…………………………………………………………………………167

4.4 Evolution of the cation exchange capacity (CEC), base saturation (%), and

aluminium saturation (%) at three depths and over the 11-month

period………………………………………………………………………....162

4.5 Evolution of nutrients in three depths and over the 11-month

period…………………………………………………………………..……..168

4.6 Limiting factors for plant production in depth 0-15cm……………………...169

4.7 Harvested biomass of Brassica juncea L………………………………….....171

4.8 Principal component analysis (PCA) of the soil

samples…………………………………………………………………….…172

5. Conclusions……………………………………………………………………….174

6. References………………………………………………………………………...175

6. 2 Anexo II Comparison of the effects of compost versus compost and biochar on the recovery of a mine soil by improving the nutrient content………...………………….183

Abastract…………………………………………………………………………...….183

1. Introducion……………………………………………………………………..…184

2. Materials and Methods…………………………………………………………....186

Índice

2.1 Soil sampling………………………………………………………………....186

2.2 Greenhouse experiment………………………………………………………187

2.3 Soil analysis…………………………………….……………………….……189

2.4 Plant growth and determination of metals in plant tissues…….…………..…190

2.5 Statistical analysis…………………………………………………………....190

3. Results………………………………………………………………………….…190

3.1 General characteristics of the settling pond soil (S), sand (SS), compost (C), and

biochar (B)……………………………………………………………...…….190

3.2 Evolution of the pH at the three depths and over the 11-month period……....192

3.3 Evolution of Total Carbon (TC) at the three depths and over the 11-month

period……………………………………………………………………..…..192

3.4 Evolution of Total Nitrogen (TN) at the three depths and over the 11-month

period…………………………………………………………………..……..194

3.5 Evolution of the cation exchange capacity (CEC), base saturation (%), and

aluminium saturation (%) at the three depths and over the 11-month period..194

3.6 Evolution of nutrients at the three depths and over the 11-month period……196

3.7 Limiting factors for plant production in depth 0-15 cm…………………...…199

3.8 Harvested biomass of Brassica juncea L…………………………………….200

4. Discussion………………………………………………………………………...200

4.1 Evolution of the pH at the three depths and over the 11-month period…..…..200

4.2 Evolution of the Total Carbon (TC) at the three depths and over the 11-month

period………………………………………………………………..….…….201

4.3 Evolution of the Total Nitrogen (TN) at the three depths and over the 11-month

period………………………………………………………………………....201

-1 4.4 Evolution of the cation exchange capacity (cmol(+)kg ), base saturation (V%),

and aluminium saturation (Al%) at the three depths and over the 11-month

period………………………………………………………………………...202

4.5 Evolution of nutrients at the three depths and over the 11-month period……203

Índice

4.6 Limiting factors for plant production in depth 1……………………………..205

4.7 Harvested biomass of Brassica juncea L…………………………………….206

4.8 Principal component analysis (PCA) in the soil samples…………………….207

5. Conclusions…………………………………………………………………...…..209

6. References………………………………………………………………………...210

6. 3 Anexo III Using compost and technosol to decrease the bioavailable metal concentration in soil from a copper mine settling pond……………………………………219

Abstract…………………………………………………………………………….….219

1. Introduction…………………………………………………………………...…..220

2. Materials and Methods…………………………………………………………....222

2.1 Soil sampling………………………..………………………………………..222

2.2 Greenhouse experiment………………………………………………………223

2.3 Soil analysis……………………………………………………………….….224

2.4 Plant growth and determination of metals in plant tissues………………...…224

2.5 Statistical analysis……………………………………………………………225

3. Results…………………………………………………………………………….226

3.1 General characteristics of the settling pond soil (S), compost (C), technosol (T),

and biochar (B)……………………………………………………………….226

3.2 Evolution of the pseudototal concentrations of Cu, Pb, Ni, and Zn at three

depths and over the 11-month period………………………………………...227

3.3 Evolution of CaCl2-extractable (Phytoavailable) contents of Cu, Pb, Ni, and Zn

at three depths and over the 11-month period………………………………..228

3.4 Harvestable amounts of Cu, Pb, Ni, Zn and determination of metals in plant

tissues: translocation factor (TF) and transfer coefficie(TrC)……………….230

3.4.1 Harvestable amounts of Cu, Pb, Ni, and Zn in Brassica juncea L..…230

3.4.2 Translocation factor (TF)……………………………………………232

3.4.3 Transfer coefficient (TrC)………………………………………...…232

Índice

4. Discussion………………………………………………………………………...233

4.1 Evolution of the pseudototal contents of Cu, Pb, Ni, Zn at three depths and over

the 11-month period………………………………………………………….233

4.2 Evolution of the CaCl2-extractable (Phytoavailable) concentrations of Cu, Pb,

Ni, Zn at three depths and over the 11-month period………………………...235

4.3 Uptake and transfer of metals to Brassica Juncea L. plants……………….…237

4.3.1 Harvestable amounts of Cu, Pb, Ni, and Zn………………………....237

4.3.2 Transfer coefficient (TrC)…………………………………………...238

4.3.3 Translocation factor (TF)…………………………………………....239

5. Conclusions……………………………………………………………………….239

6. References………………………………………………………………………240

6. 4 Anexo IV Comparative effect of compost and technosol enhanced with biochar on the fertility of a mine soil……………………………………………..…………………..249

Abstract………………………………………………………………………………..249

1. Introduction……………………………………………………………………….250

2. Materials and Methods……………………………………………………………251

2.1 Experimental site, soil sampling and amendments…………………..……….251

2.2 Greenhouse experiment………………………………………………………253

2.3 Soil analyses………………………………………………………………….254

2.4 Plant growth…………………………………………………………………..255

2.5 Statistical analysis…………………………………………………………....255

3. Results…………………………………………………………………………….255

3.1 General characteristics of settling pond soil (S), compost (C), technosol (T) and

biochar (B)……………………………………………………………………255

3.2 Evolution of the pH and the cation exchange capacity (CEC)……………….256

3.3 Evolution of Total Carbon (TC) and Nitrogen (TN)……………………..…..257

3.4 Evolution of nutrients………………………………………………………...259

Índice

3.5 Harvested biomass of Brassica juncea L…………………………….………260

4. Discussion………………………………………………………………………...261

4.1 Effect of compost and technosol enhanced with biochar on soil carbon and

nitrogen concentrations……………………………………………………….261

4.2 Effect of compost and technosol enhanced with biochar on other nutrients…262

4.3 Principal component analysis (PCA) of the samples………………………....262

5. Conclusions……………………………………………………………………….264

6. References………………………………………………………………………...265

7 Discusión general……………………………………………………………….….271

7.1 Efecto de la adición de tecnosol y biochar y de la revegetación con Brassica juncea

L. sobre el suelo de la balsa de decantación de la mina de Touro (Capítulo 1- Anexo I)…..…271

7.1.1 Evolución del pH………………………………………………………...271

7.1.2 Evolución del contenido total de carbono total (CT)…………………….272

-1 7.1.3 Evolución de la capacidad de intercambio catiónico (CEC) (cmol(+)kg ),

saturación de bases (V%), y saturación de aluminio (Al%)………….….273

7.1.4 Evolución del contenido total de nitrógeno (NT) en la profundidad 0-15

cm………………………………………………………………………...274

7.1.5 Relación carbono y nitrógeno (C/N) en la profundidad 15 cm…………..275

7.1.6 Factores limitantes para la producción vegetal en la profundidad 0-15

cm………………………………………………………………………...276

7.1.7 Evolución de la biomasa cosechada de las Brassia juncea L. cultivadas..277

7.1.8 Evolución de las concentraciones fitodisponibles de Cu, Pb, Ni y Zn.….278

7.1.9 Evolución de los contenidos cosechables de Cu, Pb, Ni, Zn, Coeficiente de

Trasferencia (TrC) y Factor de Translocación (TF)……………………..279

7.1.9.1 Contenidos cosechables de Cu, Pb, Ni, Zn………………………..279

7.1.9.2 Evolución del Coeficiente de tranferencia (TrC) en las Brassica

juncea L. cultivadas sobre STP y STBP…………………………..280

Índice

7.1.9.3 Evolución del Factor de translocación (TF) en las Brassica juncea L.

cultivadas sobre STP y STBP……………………………………..280

7.2 Efecto de la adición de compost y biochar y de la revegetación con Brassica juncea L. sobre el suelo de la balsa de decantación de la mina de Touro (Capítulo 2-Anexo

II)………………………………………………………………...…………………………….281

7.2.1 Evolución del pH…………………………………………………………...281

7.2.2 Evolución del contenido total de carbon (CT)……………………………...282

-1 7.2.3 Evolución de la capacidad de intercambio catiónico (CEC) (cmol(+)kg ),

saturación de bases (V%) y saturación de aluminio (Al%)………………...283

7.2.4 Evolución del contenido total de nitrógeno (NT) en la profundidad 0-15

cm………………………………………………………………………...…284

7.2.5 Relación carbono y nitrógeno (C/N) en la profundidad 15 cm …………….276

7.2.6 Factores limitantes para la producción vegetal en la profundidad 0-15

cm…………………………………………………………………………...285

7.2.7 Evolución de la biomasa cosechada de las Brassica juncea L.

cultivadas…………………………………………………………………...286

7.2.8 Evolución de las concentraciones fitodisponibles de Cu, Pb, Ni, Zn……....287

7.2.9 Evolución del Coeficiente de tranferencia (TrC) y Factor de translocación

(TF) en las Brassica juncea L. cultivadas sobre SCP y SCBP……………..290

7.3 Comparativa del efecto de tratamientos elaborados con compost y biochar frente a la combinación de tecnosol y biocha, ambos vegetados con Brassica juncea L. (Anexo III-

IV)……………………………………………………………………………………………...290

7.3.1 Evolución del pH…………………………………………………………...... 290

7.3.2 Evolución del contenido total de carbon (CT)……………………………...... 291

-1 7.3.3 Evolución de la capacidad de intercambio catiónico (CEC) (cmol(+)kg )..….292

7.3.4 Evolución del contenido total de nitrógeno (NT) en la profundidad 0-15

cm………………………………………………………………………….…294

Índice

7.3.5 Relación carbono y nitrógeno (C/N) en la profundidad 15 cm………………294

7.3.6 Evolución del contenido en nutrientes………………………………………..295

7.3.7 Evolución de la biomasa cosechada de las Brassica juncea L. cultivadas…...296

7.3.8 Evolución de las concentraciones fitodisponibles de Cu, Pb, Ni, Zn………...296

7.3.9 Evolución de las concentraciones de Cu, Pb, Ni y Zn en el agua de poro a lo

largo de las tres profundidades……………………………………………….299

7.3.9.1 Evolución de la concentración de Cu en el agua de poro a lo largo de las

tres profundidades………………………………………………………..300

7.3.9.2 Evolución de la concentración de Pb en el agua de poro a lo largo de las tres

profundidades…………………………………………………………….302

7.3.9.3 Evolución de la concentración de Ni en el agua de poro a lo largo de las tres

profundidades…………………………………………………………….304

7.3.9.4 Evolución de la concentración de Zn en el agua de poro a lo largo de las tres

profundidades…………………………………………………………….306

7.3.10 Evolución del Factor de translocación (TF) y el Coeficiente de transferencia

(TrC) en las Brassica juncea L. cultivadas sobre STBP y SCBP……………308

8. Conclusiones………………………………………………………………...…….311

9. Bibliografía……………………………………………...... ….315

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Introducción

Índice

Introducción

1. Introducción

1.1 Degradación del suelo y contaminación

Se considera que el continente europeo presenta una marcada limitación de recursos minerales. Sin embargo, uno de los recursos más valiosos que la humanidad ha tenido es el suelo. A pesar de que más del 95% de los alimentos producidos para los seres humanos y los animales en la Tierra depende de los suelos, el suelo sigue siendo un recurso infravalorado (Panagos et al., 2016). En el año 2015, un 33% de la superficie de la Tierra se vio afectada por algún tipo de degradación (Lal, 2015) (Figura 1). Esta degradación, además de afectar negativamente al medioambiente y a la producción agrícola, también puede frenar el desarrollo, especialmente en los países donde la agricultura es el motor para potenciar su economía. De hecho, según la FAO (2015) se define la degradación de un suelo como un cambio en el estado de salud del suelo dando como resultado una disminución de la capacidad del ecosistema para proporcionar bienes y servicios a sus beneficiarios. La degradación del suelo, además de provocar impactos ambientales y económicos, también puede afectar a la salud humana, por ejemplo, mediante la contaminación del suelo y agua por elementos potencialmente tóxicos (EPT). El coste anual de remediar las zonas degradadas o contaminadas en la Unión Europea sería de 17,3 billones de euros según las estimaciones de Tóth et al. (2016). Estos datos están corroborados por informes realizados en 2015 por la Agencia Europea del Medio Ambiente (AEMA), según la cual los costes de los proyectos de remediación suelen oscilar entre 50.000 y 500.000 euros, mientras que zonas muy amplias pueden requerir incluso más de 5 millones de euros.

Debido al problema global en el que se ha convertido la degradación del suelo, la 68ª Asamblea General de la ONU (ONU, 2013) le dio una importancia crucial a la conservación suelo declarando:

- La sostenibilidad de los suelos es clave para hacer frente a las presiones de una población creciente.

- La gestión sostenible de los suelos puede contribuir a suelos sanos y, por lo tanto, a la seguridad de los alimentos y a ecosistemas estables y sostenibles.

- La buena gestión del suelo es importante a nivel económico y social, en particular por su contribución al crecimiento económico, la biodiversidad, la agricultura sostenible y la seguridad alimentaria, que a su vez son fundamentales para erradicar la pobreza y permitir la mejora de las condiciones de las mujeres en algunos países.

- Es urgente abordar temas como el cambio climático, la disponibilidad de agua, la desertificación, la degradación del suelo y la sequía, ya que se plantean como desafíos globales.

- Existe una necesidad urgente, a todos los niveles, de sensibilizar y promover el uso

Introducción sostenible los recursos limitados del suelo usando la mejor información científica disponible y aprovechando todas las dimensiones del desarrollo sostenible.

Figura 1. Mapa de Degradación del Suelo (Rekacewicz, 2012).

La contaminación del suelo es una forma química de degradación y una de las principales preocupaciones a nivel mundial debido a sus efectos adversos sobre la salud de los ecosistemas y la seguridad alimentaria. Existen varios orígenes de dicha contaminación como los vertederos controlados e incontrolados, los vertidos accidentales, la minería, la fundición de minerales metalíferos, la aplicación de lodos de depuradora a suelos agrícolas, etc. Estos diversos orígenes de la contaminación son los responsables, entre otros, de la migración de contaminantes a zonas no contaminadas mediante lixiviados o partículas en suspensión, contribuyendo a la contaminación de los ecosistemas (Ghosh y Singh, 2005, Rinklebe et al., 2016). Se ha estimado que, el número aproximado de zonas potencialmente contaminadas en Europa, es de unos 2,5 millones, de las cuales aproximadamente 342.000 necesitan una rápida remediación (Wcisło et al., 2016)

Por otro lado, el aumento de la presión demográfica y de la demanda de servicios sobre la Tierra, también amenazan la calidad y las funciones de regulación de los recursos naturales del suelo, agua y aire de los que depende la sostenibilidad del suelo (Dumanski y Pieri, 2000). Un claro ejemplo de esto es lo que ocurre en la India donde vive el 18% de la población mundial y el 15% del ganado, pero sólo tiene el 2,4% de la superficie terrestre (Gomiero, 2016). La ocupación de suelos también acentúa la degradación del mismo. Un ejemplo son las explotaciones mineras. Generalmente, este tipo de degradación produce una pérdida irreversible de suelo y suele terminar en la contaminación del mismo. Según la Ley 22/2011, de 28 de Julio, de residuos y suelos contaminados, en su Artículo 3 apartado X, se define suelo contaminado como todo aquel cuyas

Introducción características físicas, químicas o biológicas han sido alteradas negativamente por la presencia de componentes de carácter peligroso de origen humano, en concentración tal que comporte un riesgo para la salud humana o el medio ambiente, de acuerdo con los criterios y estándares que se determinen por el Gobierno.

Según estudio realizado por Van Liedekerke et al. (2014) para la Unión Europea, la contaminación por metales es una de las más importantes en la actualidad. Este tipo de contaminación afecta de manera importante a la calidad del suelo y amenaza seriamente todo el ecosistema circundante, ya que los recursos de aguas superficiales y subterráneas, la flora, la fauna, la salud humana e incluso la calidad del aire pueden verse afectados. Las fuentes más importantes de este tipo contaminación son la minería (Figura 2), la generación de energía a partir de combustibles fósiles, la metalúrgica, la electrónica, las industrias químicas, las actividades agrícolas y el vertido/incineración de residuos. Entre estas actividades, la minería es una fuente muy contaminante, liberando altos niveles de metales. En tales circunstancias, los suelos resultan altamente degradados en sus funciones ecológicas (ciclo de nutrientes, almacenamiento de agua, hábitat microbiano, apoyo al crecimiento de las plantas, etc.) (Abad-Valle et al., 2017). En España hay reconocidas 71.202 zonas contaminadas, de las cuales 285 zonas lo estarían por metales e hidrocarburos aromáticos (Van Liedekerke et al., 2014)

Figura 2. Impactos de una explotación minera a cielo abierto (Mina de Touro, A Coruña).

1.2 Minería de superficie

La minería es la actividad industrial dedicada a la obtención de georrecursos para el abastecimiento de materias primas a la población. Es evidente que no se puede prescindir de la explotación de los recursos minerales y que esta actividad probablemente se intensificará en el futuro. La minería a cielo abierto se basa en la extracción de yacimientos poco profundos en los que se elimina la capa superficial para conseguir llegar al material de interés y extraerlo. Este tipo de minería provoca un gran impacto ecológico, social y cultural. Además, es una actividad industrial no sostenible, ya que la explotación del recurso supone su agotamiento y la recuperación

Introducción total de la zona explotada es prácticamente imposible. Debido a estas características, la minería de superficie es una actividad que origina suelos muy difíciles de recuperar (Instituto Geológico y Minero de España, 2004).

En la figura 3 se muestra un ejemplo gráfico del proceso de extracción del cobre desde la mena hasta su concentración en forma de cátodo con un 99% de pureza (proceso que se llevaba a cabo en la mina de Touro (Galicia, España)).

Figura 3. Proceso de extracción del cobre desde la mena hasta su concentración en forma de cátodo (Asensio, 2013).

El material de la mena se extrae con explosivos o maquinaria y se reduce de tamaño (pasos 1 y 2 de la Figura 2). Luego se realiza un proceso para concentración del metal, para lo cual hay varios métodos. Uno de ellos, la flotación, es en el cual se transporta el material de la mena que ya es de pequeño diámetro, como un lodo, a una balsa de flotación (paso 3). En la balsa se añaden productos químicos al lodo para que el metal que se quiere beneficiar se adhiera a burbujas de aire. Cuando el aire es forzado a pasar a través del lodo, las burbujas que suben

Introducción arrastran con ellas las partículas del metal. Se forma una capa de espuma cargada de metal en la superficie de la balsa de flotación (paso 4), la cual luego se recoge, se somete a un proceso de electrodeposición para separar el cobre (paso 5) y finalmente el metal se concentra para obtener un cátodo de elevada pureza (paso 6). En el fondo de la balsa de flotación queda un lodo con el material que no ha sido aprovechado. Como se puede observar, en la minería metálica, los materiales de desecho se acumulan en dos sitios distintos dependiendo de su tamaño de partícula. El material con diámetro mayor de 1 mm se deposita en las escombreras, mientras que el material fino producido por decantación en la balsa de flotación se acumula en la propia balsa o en otro lugar (Figura 4). Un tiempo después de que la actividad minera haya finalizado, la balsa está seca. Según la FAO en el año 2006 tanto las balsas de decantación secas como escombreras de las minas han sido oficialmente aceptadas como suelos, ya que tienen propiedades y pedogénesis dominadas por su origen técnico.

1.2.1 La mina de Touro

La mina de Touro está situada en el ayuntamiento de Touro localizado al sur de la provincia de A Coruña y limitando con el río Ulla (42º52´34´´N, 8º20´40´´W) (Figura 4). Esta zona pertenece al grupo más importante de mineralizaciones de cobre de Galicia, asociado al macizo básico de Santiago. Está formado casi exclusivamente por anfibolitas, que en algunas localidades aparecen en facies granulita y esquistos verdes. Estas anfibolitas poseen un alto contenido en granate y otros minerales metálicos. Forman parte de un gran arco de esquistos y anfibolitas de composición básica que rodean la villa de Ordes, dentro de los dominios de rocas máficas y relacionadas (Vega et al., 2005).

Figura 4. Mina de Touro.

Este yacimiento fue explotado por Explosivos Río Tinto S.A. y su cubicación se calcula en más de 25 millones de toneladas, con una ley de cobre de alrededor del 0,63% (IGME, 1982).

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Se extrajo calcopirita y pirrotina entre los años 1973 y 1988 definiendo las cortas de Bama, Brandelos, Arinteiro y Vieiro. Desde hace ya bastantes años, la empresa Explotaciones Gallegas obtiene material para la capa de rodadura de carreteras, tanto directamente de la roca como de las escombreras de las antiguas explotaciones, con lo que las escombreras han aumentado extraordinariamente de tamaño. Estas escombreras están constituidas por la roca fácilmente oxidable que no es adecuada para la utilización mencionada (Asensio, 2013). Están formadas por grandes pilas de gravas con fuertes pendientes y ocupan un área de 760 ha. Estas escombreras no comenzaron a ser restauradas hasta el año 2002 (Vega et al., 2004). Sin embargo, esta recuperación es sólo parcial, por lo que siguen causando un fuerte impacto negativo. En la mina de Touro, la riqueza en sulfuros de hierro y cobre en la anfibolita, junto con la exposición de una gran superficie de la misma en el material fragmentado acumulado en los taludes, dan lugar a una rápida meteorización, con oxidación de sulfuros y liberación de H+ a las zonas más próximas (Pérez-Otero, 1992). Además, la existencia de elevadas concentraciones de metales, no sólo Cu y Fe, sino también Ni, Mn, Cr, Pb y Zn, junto con la posibilidad de aumento de su solubilidad al disminuir el pH, causan una fuerte contaminación de las aguas y los suelos circundantes (Asensio et al., 2011).

1.2.2 Residuos mineros

Como ya ha sido comentado, los procesos mineros a menudo generan grandes cantidades de residuos. Estos residuos se depositan generalmente en el suelo y ocupan una gran superficie. En muchos casos, los residuos mineros se caracterizan por una alta concentración de metales y semimetales, ser sustratos muy deficientemente estructurados, poseer un bajo contenido de nutrientes y baja capacidad de retención de agua entre otros factores limitantes para la producción vegetal. Estas propiedades hacen que los residuos mineros sean susceptibles a la erosión del viento y del agua y que actúen como un foco continuo de contaminación ambiental para el entorno terrestre y los ecosistemas acuáticos (Puga et al., 2016, Zhou et al., 2015). Una problemática asociada a los suelos degradados de mina es el drenaje ácido de mina (Figura 5).

La industria minera es la mayor productora de este tipo de efluentes, en particular las minas abandonadas. Estas aguas suponen un riesgo para el medioambiente ya que suelen contener concentraciones elevadas de EPTs. El drenaje ácido de mina se forma a causa de la oxidación acelerada de pirita de hierro (FeS2) y otros minerales sulfurosos resultantes de la exposición de estos minerales al oxígeno y agua, como consecuencia de la extracción y tratamiento de minerales metálicos (Johnson y Hallberg, 2005). Esto es lo que ocurre por ejemplo en la mina de Touro (Galicia, España) (Forján et al., 2014).

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Figura 5. Drenaje ácido de mina (Mina de Touro, A Coruña).

En España, el Instituto Geológico y Minero (IGM) en el año 2002, comenzó el inventario sobre balsas de depósitos de lodos de minas con un total de 524 abandonadas y solo 54 recuperadas como en la mina de Touro (A Coruña). Este inventario se ha actualizado con el paso del tiempo y su última actualización según el IGM es del año 2015 (Figura 6A). Por otro lado, en el año 1989 el IGM también comenzó un inventario de escombreras de mina y canteras, cuya última actualización es también del año 2015 (Figura 6B). Observando estas figuras (6A, 6B), queda patente el problema de la mala gestión de la minería y el problema medioambiental actual.

Figura 6. Inventario Nacional de Depósitos de Lodos y Escombreras (IGM, 2015). A, inventario balsas de depósitos de lodos de minas. B, inventario de escombreras de mina y canteras.

1.3 Producción y gestión de residuos

Otro problema de la sociedad actual, altamente industrializada y consumista, es el uso incontrolado de productos químicos en la industria y en las actividades domésticas, lo que ha conducido, a menudo sin darse cuenta, a la grave contaminación del aire, agua y suelo. Muchos

Introducción de estos productos químicos son utilizados en la producción de alimentos, medicamentos y catalizadores para procesos industriales (Wei y Yang, 2010). Muchos de estos residuos son mal gestionados incluso siguiendo la legislación, filtrándose o desechándose, terminando de esta forma en el medio ambiente alterando los ecosistemas. Los residuos producidos por nuestras industrias incluyen sustancias tóxicas para muchas especies incluidos los seres humanos. Además, los residuos domésticos y municipales, incluyendo los lodos de aguas residuales, restos de elaboración de alimentos, desechos domésticos, materiales de construcción, productos de petróleo entre otros, se han convertido en uno de los problemas ambientales más acuciantes de nuestro tiempo (Hillel, 2008).

Para solucionar el problema de la recuperación de suelos contaminados o degradados, recientemente está aumentando la demanda de la utilización enmiendas que sean aplicables y económicas (Anawar et al., 2015, Puga et al., 2016). Dichas enmiendas han de ser acondicionadoras del suelo y tener la capacidad de inmovilizar los agentes contaminantes. Los materiales con los que se elaboren estas enmiendas deben ser abundantes, disponibles, biodegradables, y originados a partir de fuentes renovables (Rinklebe et al., 2016). Algunos de estos materiales son los residuos que se producen en la sociedad actual, como los residuos de industrias agroalimentarias (Islas-Valdez et al., 2015), residuos que actualmente también son un problema por su compleja gestión.

Gestionar residuos sin acumularlos o incinerarlos, dándoles otro tipo de salida, es un problema que trae en jaque actualmente, y cada vez más, a los gobiernos. Por ejemplo, en España en el año 2011 se elaboró la Ley 22/2011 de residuos y suelos contaminados que tiene como objetivo regular la gestión de los residuos impulsando medidas que prevengan su generación y mitiguen los impactos adversos sobre la salud humana y el medio ambiente asociados a su generación y gestión, mejorando la eficiencia en el uso de los recursos. Esta ley tiene asimismo como objeto regular el régimen jurídico de los suelos contaminados.

En España, la última serie de datos sobre residuos fue publicada el 7 de Diciembre de 2015 por el INE concernientes al año 2013. Casi la mitad de los residuos fueron vertidos o incinerados quedando patente que las políticas de revalorización de residuos no están siendo efectivas. Estos datos demuestran que la utilización de residuos para la elaboración de enmiendas puede ser una salida para los mismos. Cabe destacar también los datos publicados por el INE en el año 2014 (Tabla 2), referente a la serie de datos del año 2012, en la cual de observa que, de 42,875 millones de toneladas de residuos industriales, 22,509 millones de toneladas pertenecen a la industria minera. Además, en la Tabla 2 también se observa como el 46,4% de los residuos fueron vertidos o incinerados.

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Tabla 2. Tratamiento final de residuos año 2012. Unidad: miles de toneladas (Tn) Residuos tratados Cantidad % sobre el total %variación interanual (por tipo de gestión) Total residuos gestionados 44.864,1 100,0 10,0 No peligrosos 43.099,2 96,1 10,9 Peligrosos 1.765,9 3,9 -8,6 Reciclado 24.050,2 53,6 17,0 No peligrosos 22.733,9 50,7 18,6 Peligrosos 1.316,3 2,9 -5,9 Vertido 17.771,6 39,6 5.1 No peligrosos 17.487,0 39,0 5,8 Peligrosos 285,6 0,6 -26,4 Incineración 3.042,3 6,8 8,5 No peligrosos 2.878,3 6,4 10,9 Peligrosos 164,0 0,4 -8,6 INE 2014

Además, el correcto reciclaje de residuos de origen orgánico ayudaría a frenar las emisiones de gases de efecto invernadero. En el presente año 2017, Masullo propuso un modelo según el cual, por cada tonelada de residuos orgánicos tratados, ahorraría la emisión de 196,2 kg de CO2eq a la atmósfera (Figura 7).

Figura 7. Impacto climático de la gestión de residuos orgánicos. Los bloques rodeados de rojo representan la solución propuesta por Masullo (2017).

La generación y mala gestión de residuos constituye un grave problema ambiental. Tanto el abandono de residuos como su mala gestión provocan impactos importantes al medioambiente. Si los residuos fueran gestionados de forma correcta, éstos podrían reconvertirse en recursos (renovables) contribuyendo al ahorro en el uso de materias primas, lo cual redunda en una menor

Introducción explotación de recursos naturales y a un desarrollo sostenible.

1.4 Residuos y enmiendas

Partiendo de la base de que la producción cero de residuos es imposible, una forma de dar salida de forma integrada a una gran cantidad de residuos industriales, municipales y domésticos puede ser su reutilización como enmiendas del suelo (Macía et al., 2014, Pérez- Esteban et al., 2014, Zorzona et al., 2012). Según el informe sobre la materia orgánica de la Estrategia Europea de Protección del Suelo del año 2006, la adición de residuos adecuados y con buenas prácticas de gestión en forma de enmiendas al suelo producen mejoras significativas en las condiciones de fertilidad, estabilidad estructural, porosidad y actividad biológica de los suelos, que redundan en un mejor cumplimiento de sus funciones productivas y ambientales, con beneficios como: incrementos en las cosechas, mejora de la capacidad de depuración del agua, mayor resistencia frente a los contaminantes, incremento del secuestro y almacenamiento de carbono (Biederman et al., 2017, Pérez-Esteban et al., 2012). Con este tipo de gestión se contribuye a la valorización de los residuos en la línea establecida por la Unión Europea en la Decisión 2000/532/CE. Por otro lado, en el anexo IIB de la Directiva del Parlamento Europeo y del Consejo sobre los residuos, 2005/0281 (COD), que modifica la Directiva 75/442, se enumeran las posibles operaciones de valorización, figurando como operación R10 “el tratamiento de los suelos, produciendo un beneficio a la agricultura o una mejora ecológica de los mismos”. Además de revalorizar residuos mediante la elaboración de enmiendas, éstas tienen beneficios añadidos al ser tratamientos `ìn situ”. Las técnicas de recuperación de suelos degradados `ìn situ´´ se basan en la aplicación directa sobre el suelo contaminado de enmiendas y / o plantas y sus microorganismos asociados (Basta et al., 2001). Estas técnicas son baratas y fáciles de aplicar y mejoran la funcionalidad del suelo de manera significativa, pero son algo más lentas que las `èx situ´´ y requieren una monitorización del proceso de recuperación (Pavel et al., 2014).

Las enmiendas orgánicas utilizadas en la recuperación de suelos provienen de diferentes fuentes como la agricultura, la silvicultura, las zonas urbanas y la industria. A continuación, se detallan algunos de estos residuos y sus orígenes: los más frecuentes son los generados por la agricultura como el estiércol (Islas-Váldez et al., 2016, Pérez-Esteban et al., 2014), otros residuos empleados en enmiendas derivados de la agricultura incluyen los residuos de los cultivos. Otro tipo de residuos utilizados son los producidos por la industria maderera y derivadas los cuales son de diversa índole como lodos de destintado, astillas y virutas de madera (Pérez-Esteban et al., 2014). En el pasado, los residuos de madera de las industrias forestales y madereras eran incinerados para producir ceniza de madera. Para mantener la calidad del aire, diversos países han eliminado esta práctica, lo que significa que hay más producto en bruto que puede ser aplicado al suelo. Las enmiendas orgánicas que tienen su origen en residuos de tipo urbano incluyen biosólidos (lodos de aguas residuales, lodos municipales) (Alvarenga et al., 2016, Paradelo et al.,

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2011), y los componentes biodegradables de los residuos sólidos urbanos, los cuales pueden tener un origen diverso. Por otro lado, de la industria agroalimentaria derivan subproductos orgánicos que se pueden aplicar al suelo (Larney y Angers, 2011). Se han sugerido diversas técnicas para rehabilitar suelos con niveles peligrosos de elementos traza (Kargar et al., 2015), que abarcan enfoques de ingeniería y métodos químicos / biológicos que buscan la descontaminación o la estabilización del suelo. Algunas de estas técnicas consisten en la mezcla de los residuos mencionados anteriormente o por separado dando lugar diferentes tipos de enmiendas según su procesamiento como el compost, los tecnosoles o el biochar. La aplicación de enmiendas está considerada como la alternativa más adecuada a bajo coste para remediar los suelos degradados por las actividades mineras (Abad-Valle et al., 2017, Macía et al., 2014, Puga et al., 2015).

1.4.1 Compost

El compostaje es un proceso biooxidativo que implica la mineralización y humificación parcial de la materia orgánica, dando lugar a un producto final estabilizado, libre de fitotoxicidad y patógenos y con ciertas propiedades húmicas (Bernal et al., 2009) (Figura 8).

Figura 8. Proceso de compostaje.

Se han desarrollado múltiples métodos y sistemas de compostaje, que van desde pequeños reactores caseros utilizados por hogares individuales, a reactores de tamaño mediano utilizados normalmente por agricultores, hasta reactores grandes y de alta tecnología utilizados por productores profesionales de compost (Fischer y Glaser, 2012). Todos los procesos de compostaje adecuados pasan por cuatro etapas: (1) mesófila, (2) termófila, (3) enfriamiento y finalmente la maduración del compost (4). La duración de cada etapa depende de la composición inicial de la mezcla, su contenido en agua, aireación y cantidad y composición de las poblaciones microbianas (Neklyudov et al., 2006).

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En la Ley 22/2011 el compost viene definido como una enmienda orgánica obtenida a partir del tratamiento biológico aerobio y termófilo de residuos biodegradables recogidos separadamente. No se considerará compost el material orgánico obtenido de las plantas de tratamiento mecánico biológico de residuos mezclados, que se denominará material bioestabilizado. El compost se utilizó comúnmente para mejorar los rendimientos de los cultivos y la fertilidad del suelo. El compost contiene cantidades significativas de nutrientes valiosos para las plantas incluyendo N, P, K, Ca, Mg y S, así como una variedad de oligoelementos esenciales. Por lo tanto, en este aspecto, el compost puede definirse como un fertilizante orgánico multi- nutriente (Fischer y Glaser, 2012). Por otra parte, aumenta el contenido de materia orgánica, disminuye la densidad aparente y la erosión y aumenta la estabilidad de los agregados y la aireación. Además de todo esto, el compost fue una de las primeras enmiendas elaboradas con residuos que se utilizaron en el campo de la recuperación de suelos degradados (Anastopoulos y Kyzas, 2015, Beesley et al., 2014). Los residuos con los que se elaboran estos compost nunca escasean ya que la producción de residuos agrícolas o urbanos es constante (Alvarenga et al., 2016, Bernal et al., 2009). Los compost son ricos tanto en compuestos lábiles como en compuestos más recalcitrantes, con concentraciones adecuadas de nitrógeno y otros nutrientes (Luna et al., 2016). Los compost pueden fijar tanto contaminantes orgánicos como inorgánicos, mejorar la densidad y estructura del suelo, reducir las pérdidas por lixiviación de nutrientes, y proporcionar una rica capa superior al suelo de forma rápida (Adl, 2008, Haritash y Kaushik, 2009). Por otra parte, se promueve el reciclaje de residuos, lo que es un beneficio adicional para el medio ambiente. Algunos residuos con los que se elaboran algunos compost hacen que dichos compost tengan una difícil salida al mercado ordinario, por lo cual una salida sostenible para estos compost es aplicación en zonas degradadas con lo que se ayuda por un lado a recuperar estos suelos y por otro se evita la acumulación de estos compost.

Las principales características cualitativas del compost que deben tenerse en cuenta son las siguientes (Neklyudov et al., 2006):

- Contenido de metales, compuestos orgánicos y microorganismos patógenos

- Ratio carbono/nitrógeno (C:N), contenido de nutrientes y microelementos

- Humedad

- Tamaño de partícula

- Contenido en sales

- La apariencia, color y olor

- Grado de estabilidad

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Algunos de los residuos con los que se elaboran los diferentes tipos de compost incluyen residuos sólidos urbanos (RSU) y lodos de depuradora que tienden a tener concentraciones elevadas de metales, residuos orgánicos de plantas de celulosa de papel y subproductos orgánicos agrícolas o agroindustriales a los que muchas veces se les atribuyen malos olores (Alvarenga et al., 2016, Sáez et al., 2016). Muchos suelos contienen niveles considerables de metales de forma natural por lo que el aporte de metales por parte de las enmiendas elaboradas con residuos muchas veces no es significativo. De hecho, la retención de metales provocada por la materia orgánica aportada reducirá la biodisponibilidad, niveles y lixiviación de elementos potencialmente tóxicos en la zona (Adl, 2008). Un problema atribuible al compost es la corta duración de su influencia en los suelos donde se aplica. Esto ha sido demostrado por autores como Walker et al. (2004) en un experimento realizado con suelo del desastre ecológico que tuvo lugar en Aznalcollar, en donde se había roto el muro de contención de la balsa de lodos, lodos ricos en As, Cd, Cu, Pb, Sb, Tl y Zn. En dicho experimento se trató este suelo con dos enmiendas, la primera enmienda consistía en un compost y la segunda en estiércol de vaca fresco. En los suelos tratados con el compost, a partir del día 31 esta enmienda perdía efecto y los contenidos de metales extraídos con

CaCl2 aumentaban, en cambio en el suelo tratado con estiércol de vaca el efecto era a más largo plazo y cuando aumentaban los contenidos extraíbles con CaCl2 lo hacían en menor cantidad. Debido a las limitaciones del compost como enmienda se dio un paso más a la hora de elaborar este tipo de tratamientos dando lugar a los tecnosoles (suelos a la carta).

1.4.2 Tecnosoles

Los Tecnosoles comprenden un nuevo grupo de suelos de referencia (GSR) y combinan suelos cuyas propiedades están originadas por su origen técnico. Presentan un contenido significativo de artefactos (algo en el suelo reconociblemente hecho o extraído de la Tierra por el hombre), o están sellados por roca dura técnica (material duro creado por el hombre, que tiene propiedades diferentes a la roca natural). Incluyen suelos de desechos (rellenos, lodos, escorias, escombros o desechos de minería y cenizas), pavimentos con sus materiales subyacentes no consolidados, suelos con geomembranas y suelos construidos en materiales hechos por el hombre (FAO, 2007). La reutilización de residuos para la elaboración de tecnosoles es una alternativa contemplada por Dirección General de Calidad y Evaluación Ambiental de la Xunta de Galicia (ITR, 2005). Esta ITR regula la elaboración de los suelos reciclados a partir de residuos como una alternativa de valorización en consonancia con las directrices de la Unión Europea en las que se fomenta la aparición de nuevas propuestas de valorización de los residuos (Figura 9). En particular, la operación de valorización R10: tratamiento de suelos, produciendo un beneficio para la agricultura o una importante mejora ecológica de éstos (ITR, 2005).

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Figura 9. Planta de elaboración de tecnosoles de la empresa TEN.

Los tecnosoles pueden producir otros beneficios adicionales tales como:

- Ser sustitutivos de materiales de interés ambiental, como las turbas o la tierra vegetal extraída de otros suelos.

- Disminución de las emisiones de gases de efecto invernadero (CO2, CH4 y NOx) producidas en la gestión de residuos por los métodos de vertido, incineración o compostaje.

- Rápida y correcta integración de diferentes elementos y sustancias en los ciclos biogeoquímicos con garantía sanitaria.

- Corrección de diferentes problemas ambientales existentes en zonas con suelos contaminados o degradados, mediante la utilización de tecnosoles de composición y propiedades adecuadas a la solución o mitigación de la situación de partida (ITR, 01/08).

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Los tecnosoles cumplen los requisitos de la estrategia de la gestión ambiental integrada. A su vez también pueden ser utilizados para la recuperación de suelos contaminados o degradados. De este modo, el tecnosol puede tener dos funciones: dar salida a los residuos y como enmienda para recuperar o mejorar suelos. Los tecnosoles derivados de residuos son una mezcla sólida de materiales naturales o sintéticos, minerales u orgánicos que, colocados en superficie, permiten la rápida integración de los componentes residuales antropogeomórficos en los ciclos biogeoquímicos así como el cumplimiento de las funciones ambientales y productivas del suelo, mejorando la situación ambiental precedente (Macías, 2004 y Macías et al., 2007). La elaboración de tecnosoles derivados de residuos (Camps et al., 2008; Yao et al., 2009), a imagen de los suelos naturales y a medida de los requerimientos de corrección de los suelos contaminados y/o degradados existentes, (“taylor made Technosols” o “Tecnosoles a la carta”), puede constituir un proceso que atienda simultáneamente a la valorización biogeoquímica de los residuos, permita la recuperación de las funciones del suelo en espacios degradados y/o contaminados y retenga una gran parte de su carbono y nitrógeno a través de su integración en los compartimentos geoquímicos de acción rápida, que son la biomasa y los suelos.

Por ello los tecnosoles podrían ser altamente beneficiosos si se utilizan en ciertos casos y suelos como (Macías et al., 2010):

- Recuperación de suelos contaminados, canteras, minas y entornos urbanos e industriales contaminados y/o degradados.

- Complemento de la tierra vegetal apilada previamente en actuaciones de restauración de suelos o vertederos.

- Sustitutivo de la turba y tierra vegetal en labores de sellado o recuperación de suelos afectados por obras urbanas, industriales, infraestructuras, etc.

- Cultivos forestales de alta intensidad (eucaliptales, choperas)

- Bosques de producción maderera.

- Suelos de cultivo en vías de degradación de sus horizontes superficiales por contaminación, empobrecimiento, pérdida de materia orgánica y fertilidad, excesivo laboreo, compactación y pérdida de estructura.

- Suelos con cultivos forzados, con alta demanda de nutrientes

Los tecnosoles no han de ser utilizados en ciertos tipos de suelos debido a los posibles riesgos medioambientales que conlleva el uso de algunos residuos. Estos suelos son (Macías et al., 2010):

- Suelos de la Red Natura.

- Suelos de áreas protegidas o de interés natural y paisajístico.

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- Suelos de elevada sensibilidad muy susceptibles de cambiar sus propiedades: turberas, marismas y marjales.

- Suelos hidromorfos (p.ej. Podsoles, Gypsisoles).

- Suelos singulares que deben ser protegidos como patrimonio edafogenético (p.ej. algunos Vertisoles, Mollisoles, Podsoles, Calcisoles, Ferralsoles, Andosoles, Ultisoles, Alfisoles rojos).

- Bosques climácicos.

- Praderas y pastizales naturales.

Un ejemplo de tecnosoles a la carta se puede observar en la figura 10 en la cual se presenta el efecto de 4 tipos de tecnosoles aplicados en la corta de Bama (Mina de Touro, Galicia). Hacia esta corta estaban dirigidas todas las aguas de escorrentía de escombreras y otras cortas aledañas. En este lugar se implantó un tecnosol de baja permeabilidad, con la finalidad de contener el agua, así como para controlar la velocidad y tiempo de residencia, permitiendo que crezca la vegetación de forma espontánea en sus laterales y regulando los flujos de caudal, al permitir el paso sólo de la capa superficial de la columna de agua. Como refuerzo del efecto controlador del pH, las aguas pasan por un tecnosol hiperalcalino, que sube el pH de las aguas hiperácidas hasta valores de entre 4,0 y 6,0, acción que hace que se precipite el Fe soluble y parte del Al y que parte de los sulfatos y metales sean adsorbidos en los precipitados de bajo grado de orden o microcristalinos inicialmente formados (Bolaños, 2014).

Figura 10. Utilización de diferentes tecnosoles para recuperar zonas con diferentes requerimientos. Macías-García et al. (2009).

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A pesar de los grandes beneficios que presentan los tecnosoles, éstos pueden tener problemas derivados de los residuos con los que se elaboran. Algunos de estos problemas son la presencia de metales y de sustancias tóxicas, la existencia de microorganismos patógenos, el exceso de nutrientes o la demanda o deficiencia de los mismos, presencia de materiales no biodegradables, salinidad, etc. Por eso es muy importante tratar este tipo de residuos antes de ser utilizados y elegir el tipo de residuo a utilizar según la zona en la que se desee aplicar. Estos problemas se ven agrandados si los tecnosoles no están bien procesados (Arbestain et al., 2008)

1.4.3 Biochar

En el año 2015, el International Biochar Initiative definió el biochar como un carbón vegetal finamente granulado de alto contenido en C orgánico y muy resistente a la descomposición. Se produce a partir de la pirólisis de plantas y de residuos. Las características de cada biochar dependen del material de partida y de las condiciones a las que se elabore (Dai et al., 2017). El biochar, producido a temperaturas entre 400 y 600 oC, tiene características orgánicas más diversificadas que los elaborados a menores o mayores temperaturas (Lehmann, 2007) (Figura 11), incluyendo estructuras alifáticas y de tipo celulosa las cuales contienen un alto número grupos funcionales de tipo C = O y C-H. Otra característica que cambia con la temperatura es el pH, cuando los productos para elaborar biochar son pirolizados a 300–399 °C el valor medio de pH es 5.01, mientras que si la pirolisis se realiza a 600–699 °C el valor medio de pH es 9.00 (Lehmann y Joseph, 2015)

Figura 11. Caraterísticas del biochar según la temperatura de pirólisis, Lehmann, (2007).

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La mayor parte del carbono que contiene el biochar presenta una estructura aromática muy recalcitrante en el medioambiente, unos altos valores de pH y capacidad de intercambio catiónica, y puede mejorar la productividad del suelo. Además, los productos secundarios derivados de su producción (aceites, gases…) pueden ser utilizados como energía verde (Rajapaksha et al., 2016, Shen et al., 2016). Como enmienda aplicable al suelo, el biochar a menudo tiene un efecto encalante, y posee una microestructura porosa que da como resultado una alta área de superficie específica y grupos funcionales activos en su superficie, lo que implica una alta capacidad de complejar metales en su superficie (Beesley y Marmiroli, 2011; Lu et al., 2012).

Figura 12. Imagen de biochar obtenido a partir de biomasa de eucalipto a 360 ºC. La imagen se obtuvo mediante un microscopio electrónico de barrido por emisión de campo (FE-SEM) equipado con un espectrómetro dispersivo de energía (EDS).

Cuando el biochar es aplicado al suelo puede aumentar la capacidad de intercambio catiónico y el pH, y a su vez, la atracción electrostática entre cationes metálicos y las partículas del suelo se hará más fuerte. El biochar puede estabilizar los metales en los suelos contaminados, mejorar la calidad de suelos contaminados y reducir de forma significativa la absorción de metales por las plantas. Por lo tanto, la aplicación de biochar puede proporcionar, potencialmente, una nueva solución para la remediación de suelos contaminados por elementos potencialmente tóxicos (EPTs) y puede mejorar la productividad de los mismos (Karer et al., 2015, Paz-Ferreiro et al., 2014, Zhang et al., 2013). La reducción de la biodisponibilidad de los EPTs y otras modificaciones en el suelo inducidas por la aplicación de biochar pueden ser beneficiosas para el establecimiento de una cubierta vegetal en la parte superior de los residuos o enmiendas para conseguir una fitoestabilización de larga duración (Puga et al., 2015). Características del biochar como su alto contenido en fracción fina y estructura porosa afectan principalmente al agua y aire de la capa

Introducción superficial del suelo. Se ha observado que la densidad aparente disminuye, aumenta la capacidad de retención de agua y se acelera la permeabilidad (Marousek et al., 2017). Además, el biochar crea un reservorio de carbono recalcitrante en el suelo ya que es C-negativo, siendo un sumidero neto de dióxido de carbono almacenado en la fracción de carbono del suelo altamente recalcitrante. El biochar también posee una elevada capacidad de retención de nutrientes, con lo que no sólo reduce las necesidades totales de fertilizantes, sino que minimiza el impacto climático y ambiental de los suelos de cultivo. Todo esto ha sido demostrado en investigaciones recientes (Biederman y Harpole, 2013, Madiba et al., 2016) en las cuales se concluyó que el biochar tiene una gran importancia a la hora de aumentar el almacenamiento de carbono en el suelo, incrementando la fertilidad del suelo, ayudando a mantener el equilibrio de los ecosistemas del suelo, y actuando como abono para el suelo o enmienda para aumentar el rendimiento del cultivo y crecimiento de las plantas mediante el suministro y la retención de nutrientes. El aumento de la fertilidad del suelo se ha atribuido a la alta área superficial del biochar (200–300 m2/g) y alta capacidad de intercambio catiónico (27,7–222,4 mmolc/kg), que aumenta la capacidad de retención de agua, absorción de nutrientes y mejora el crecimiento de plantas mediante el suministro y la retención de nutrientes.

Además de estas características positivas del biochar, autores como Fowles (2007) o Kammann et al. (2015) demostraron que la combinación del biochar con enmiendas elaboradas con residuos como el compost o los tecnosoles mejora la efectividad de los mismos y palía los defectos como la problemática de estas dos enmiendas con los EPTs (por ejemplo, metales derivados de los residuos con los que se elaboran). Un ejemplo de ello fue demostrado por Sáez et al. (2016), los cuales diseñaron un experimento para intentar solucionar la problemática de las concentraciones fitodisponibles de metales en los compost elaborados con purín de cerdo. Para ello mezclaron el compost, que contenía purín de cerdo, con biochar por un lado y con fibras de coco por otro. Los resultados obtenidos mostraron que las mezclas de compost y biochar presentaban concentraciones fitodisponibles de metales más bajas que en el compost solo o el compost y fibras de coco.

Además, el biochar puede actuar como agente estructurante en el proceso de compostaje y regulando la humedad de la enmienda, también como fuente de carbono y energía para los microorganismos. Ha sido comprobado que, al aplicar biochar en el compostaje de estiércol de aves de corral, se optimizó el proceso de compostaje mediante la reducción de las emisiones de olor y la pérdida de N, así como la producción de compost más equilibrado en su composición de nutrientes (Adhikari et al., 2009, Bulter et al., 2001, Dias et al., 2010). Por todo ello, el biochar tiene una función doble que sería por un lado el reciclaje de residuos y, por otra, la mejora de suelos o enmiendas donde éste sea aplicado (Figura 13).

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Figura 13. Relación entre el biochar, el reciclaje de residuos y mejora del suelo. Imagen modificada de Tang et al. (2013).

Una característica poco conocida del biochar es su capacidad a la hora de aumentar la resistencia de los cultivos a enfermedades, como agente de control de enfermedades en la agricultura, y por consiguiente al aumento de la productividad de los cultivos (Tang et al., 2013). Elad et al. (2010) encontraron que la gravedad de las enfermedades foliares creadas por Botrytis cinerea Pers. Fr. y Oidiopsis sicula Scalia en los cultivos de tomate y pimienta se redujeron significativamente cuando el biochar era aplicado al suelo. Otro experimento relacionado con este beneficio del biochar fue el llevado a cabo por Harel et al. (2012), quienes exploraron la capacidad del biochar elaborado con astillas y residuos provenientes de cultivos de invernadero para inducir resistencia sistémica en plantas de fresa contra B. cinerea, Colletotrichum acutatum y Podphaera aphanis, para lo cual examinaron algunos de sus impactos en los mecanismos de defensa de las plantas a nivel molecular por PCR en tiempo real cuantificando la expresión relativa de 5 genes relacionados con la defensa de las plantas (FaPR1, Faolp2, Fra as, Falox y FaWRKY1). Los resultados de la PCR en tiempo real sugirieron que la adición de biochar estimuló una serie de vías generales de defensa.

1.4.4 Capacidad de sorción de las enmiendas elaboradas con residuos (compost, tecnosol, biochar)

La sorción es el proceso por el cual una sustancia en disolución llega a ser retenida por la superficie de una partícula sólida. La naturaleza de la superficie del sólido y la del sorbato determinan la afinidad. Puesto que los mecanismos de retención de iones metálicos en la

Introducción superficie del suelo son frecuentemente poco conocidos, en lugar del término adsorción es preferible utilizar ``sorción´´ (Sparks et al., 1999) que, en general, implica la pérdida de los iones metálicos de una fase acuosa contigua a una sólida y engloba tres importantes procesos: adsorción, precipitación y fijación. (Apak, 2002; Vega et al., 2005).

Algunos componentes del suelo como, por ejemplo, los minerales de la arcilla, tienen una carga negativa superficial permanente (Kabata-Pendias, 2001). Iones con carga positiva, como los de los óxidos e hidróxidos de hierro y aluminio y algunas macromoléculas orgánicas, poseen grupos funcionales que cambian de carga con el pH, por lo que los iones metálicos pueden llegar a ser retenidos en ellas a través de reacciones químicas de complejación o por atracción electrostática (Alloway, 1995; Kabata-Pendias, 2001). Diversas enmiendas elaboradas con residuos (compost, tecnosoles, biochar) ayudan a retener tanto los metales que contienen los elementos con los que se elabora, como los que hay en el suelo donde se aplica.

En el año 2015, Anastopoulos y Kyzas realizaron un artículo de revisión donde exponían que el compost, además de realizar un papel como enmienda del suelo, también tiene un papel importante como biosorbente debido a su capacidad de sorción. Estos autores revisaron diferentes experimentos sobre diferentes compost y metales. Otro ejemplo, de la capacidad de sorción del compost, fue expuesto por Smith (2009), el cual propuso que existían numerosas y buenas pruebas experimentales que demuestran la reducción de la biodisponibilidad y la captación de metales por los cultivos era menor en los suelos donde se aplicaron biosólidos compostados en comparación con otros tipos de residuos sin compostar como lodos de depuradora. Por todo ello, es probable que los procesos de compostaje contribuyan a reducir la biodisponibilidad de metales en los suelos enmendados en comparación con otras técnicas como la aplicación directa de algunos residuos. Otros autores como Vaca-Paulín et al., (2006) demostraron la capacidad del compost a la hora de incrementar la capacidad de sorción de Cu y Cd una vez aplicado al suelo.

Los tecnosoles son enmiendas que también suelen aumentar la capacidad de sorción de metales en los suelos en los que son aplicados. Un ejemplo de esta mejora de la capacidad de sorción una vez aplicado un tecnosol fue la estudiada con detalle por Vega et al. (2009), para lo cual se utilizaron tres muestras de suelo de la mina de Touro (A Coruña) sin enmendar y otras tres muestras suelo de la misma mina enmendado con un tecnosol durante 2 años. Al final del experimento concluyeron que el tecnosol provocó en el suelo un aumento significativo en su capacidad de sorción de Cd, Cu y Pb.

El biochar es otra de las enmiendas con gran capacidad de sorción de metales. Park et al. (2013), utilizando dos biochars de origen distinto y con los resultados de los estudios de cinética y las isotermas de sorción, concluyeron que el biochar es útil en la eliminación de metales tanto en el suelo como en el agua. El material de partida y temperatura a la que se elabora el biochar es

Introducción un factor crucial en cuanto a su capacidad de sorción. Si se incrementa la temperatura de pirólisis, incrementa el área superficial del biochar, la cual facilita la inmovilización de metales (Tang et al., 2013). Esta capacidad de sorción de metales no se limita solo a los metales, sino también a contaminantes de tipo orgánico, lo cual ha sido demostrado por diferentes autores (Oleszczuk et al., 2012 y Wang et al., 2010). Por último, cabe destacar la capacidad de sorción del biochar en cuanto a los pesticidas lo que lo hace una enmienda muy completa (Tang et al., 2013)

1.5 Fitorremediación

La fitorremediación es el uso de plantas y microorganismos asociados al suelo para eliminar o reducir los contaminantes en las diferentes matrices mediombientales (aire, suelo y agua) (Compant et al., 2010, Rascioa y Navari-Izzo, 2011). Es una tecnología amable con el medioambiente y que se puede utilizar para extraer o inmovilizar metales, metaloides, radionucleidos y así como xenobióticos orgánicos. Los bajos costes de la fitorremediación son una ventaja en comparación con las tecnologías convencionales junto a su menor impacto ambiental. Por lo tanto, la fitorremediación tiene un alto potencial, debido a su relación coste- efectividad es altamente rentable, es ecológica ya que se mantiene con energía solar. La fitorremediación utiliza procesos químicos, físicos y biológicos para eliminar, degradar, transformar, o estabilizar los contaminantes presentes en el aire, suelo y agua. Además, mediante estos procesos, la movilidad y la biodisponibilidad de los metales y su entrada en la cadena alimentaria se reduce considerablemente (Chirakkara et al., 2016, Kushwaha et al., 2016, Pinto et al., 2015).

1.5.1 Fitoestabilización

La fitoestabilización reduce el riesgo intrínseco planteado por los contaminantes sin eliminarlos del lugar al reducir su biodisponibilidad en el suelo. Los suelos contaminados normalmente presentan una cobertura vegetal poco estable o directamente ésta está ausente debido a los efectos tóxicos de los contaminantes o a las perturbaciones físicas. Una solución simple para la estabilización de los metales es la revegetación con especies o poblaciones de plantas tolerantes a metales, muy útil para la estabilización de residuos mineros. Esta técnica impide la erosión y lixiviación. Se podría definir como una prevención parcial de la transferencia del contaminante desde un suelo contaminado a zonas adyacentes o aguas subterráneas. Implica una estabilización mecánica del suelo por las raíces de las plantas, protege la superficie del suelo contra la erosión así como reduce la infiltración a aguas subterráneas al mejorar la transpiración (Clemente et al., 2004). En nuestro caso, esta vegetación ayudaría a fijar las enmiendas empleadas como tecnosoles, compost o biochar, fuente de nutrientes. Esto es importante ya que, en las zonas contaminadas, además de los problemas de toxicidad existen problemas de déficit de nutrientes,

Introducción lo cual es una limitación importante para la revegetación. En el caso de residuos mineros, el problema más común es la deficiencia de nitrógeno y, por otro lado, los valores extremos de pH (Pérez-Esteban et al., 2012). Algo muy importante que aporta la revegetación es que, una vez establecida, la nueva cubierta vegetal puede actuar como un sistema auto-sostenible. Las plantas no solo producen materia orgánica que se recicla en el suelo, sino que también pueden mejorar las condiciones microclimáticas, la estructura del suelo, proteger el suelo de la compactación, erosión y aumentar la actividad biológica (Clemente et al., 2004). En resumen, los objetivos de la fitoestabilización son:

- Reducir el riesgo que presenta un suelo contaminado por la disminución de la biodisponibilidad de metales utilizando una combinación de plantas y enmiendas (Inmovilización / inactivación). - Su objetivo no es ser una tecnología para la limpieza de suelos contaminados, pero si para la estabilización (inactivación) de elementos traza que sean potencialmente tóxicos. - La contaminación se 'inactiva' en la zona donde se localiza para prevenir su dispersión.

Para que un suelo degradado, como por ejemplo un suelo de mina, se considere que está recuperado es necesario que pueda sostener una cubierta vegetal estable. En los suelos degradados debido a sus malas condiciones se suele instaurar una primera cubierta vegetal con plantas fitoestabilizadoras (Frérot et al., 2006) a la vez que se lleva a cabo la aplicación de enmiendas (compost, tecnosol, biochar) que mejoren las condiciones limitantes de dichos suelos (Figura 14).

Figura 14. Diferencia entre un suelo contaminado por metales sin tratar frente a otro contaminado y tratado con enmiendas y plantas fitoestabilizadoras. Imagen modificada de Ruttens et al. (2006).

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Una cubierta vegetal consistente en plantas fitoestabilizadoras, además de estabilizar los metales es muy eficaz a la hora de reducir de la erosión superficial porque las raíces penetran en el suelo y lo estabilizan. Además, la vegetación puede devolver una gran proporción de percolación de agua a la atmósfera a través de la transpiración, lo que reduce las concentraciones de metales solubles que entrarían en los cursos de agua. Una cubierta vegetal también reduce el impacto visual causado, por ejemplo, en el paisaje por las explotaciones mineras. Una revegetación exitosa puede permitir el uso recreativo de nuevo de la zona recuperada, e incluso instaurar de nuevo explotaciones agrícolas o forestales, si las condiciones son favorables (Larney y Angers, 2011, Tordoff et al., 2000).

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2. Estudios previos

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2.1 Effect of amendments made of waste materials in the physical and chemical recovery of mine soil

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Journal of Geochemical Exploration 147 (2014) 91–97

Contents lists available at ScienceDirect

Journal of Geochemical Exploration

journal homepage: www.elsevier.com/locate/jgeoexp

Effect of amendments made of waste materials in the physical and chemical recovery of mine soil

R. Forján ⁎, V. Asensio, A. Rodríguez-Vila, E.F. Covelo

Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, As Lagoas-Marcosende, 36310 Vigo, Pontevedra, Spain article info abstract

Article history: The process of obtaining minerals required by society entails a series of impacts caused by mining. In order to Received 20 January 2014 minimise these impacts, amendments have now been used for several years made of waste material in order Accepted 15 October 2014 to recover mine soils. Two amendments were used in this experiment: A1 (a mixture of sludges from a bleach Available online 23 October 2014 plant, urban solid waste and material from the area around the mine) and A2 (sludge from a puriﬁcation plant, wood chips and remnants from agri-food industries). These amendments can improve the characteristics of Keywords: Organic waste-amendment the mine soils by increasing their pH and concentrations of carbon and nutrients, as well as reducing the concen- Mine soil tration of metals in bioavailable form. However, the use of wastes can add metals to soils; so it is important to Metal control their concentrations before being used. With the aim of knowing how these amendments perform Soil remediation over time, an experiment was carried out in plant pots, amending a soil from the settling pool from a former copper mine located in Touro, in north western Spain. The pseudo-total and DTPA-extractable concentration of metals, nutrients in phytoavailable form and pH of the soil were measured over a 3-month period. At the end of the experiment, it was found that both amendments caused in increase in the pH and concentrations of basic cations, carbon, and humic and fulvic acids. The amendment made of solid urban waste (SUW) caused an increase in the concentration of DTPA-extractable Pb and Zn. For these reasons, when applying these amendments it is important to take into account to which types of soils they can be applied. To amend this mine soil, the materials used in the amendments A1 and A2 would be valid, as the soil from the pool is already contaminated by metals and cannot be used for agricultural purposes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction with great potential. These types of amendments can improve the characteristics of the soils, increasing the pH and concentrations of carbon The consumption of minerals by human beings is the main cause of and nutrients, as well as reducing the concentration of metals in a bio- contamination by some trace elements in the biosphere, such as copper, available form. This helps plants to establish and encourage microbial lead or zinc (László, 2005). Mining activities cause impacts such as a loss activity. It is also a less costly technique than off-site techniques such of arable land, woodland and pasture, soil degradation and pollution of as incineration, chemical extraction using solvents, thermal desorption air and water (Xia and Cai, 2002). Plants and organisms in the soil have or biological treatment in reactors, and also does not result in sub- serious difficulties in developing in mine areas due to the low pH of the products (Brown et al., 2005; Mench et al., 2003; Pérez-de-Mora et al., soil, the lack of organic material, nitrogen and nutrients, the low capac- 2007). In turn, these organic amendments allow for the rapid inclusion ity for cation exchange, and the high concentration of heavy metals of waste produced by human beings into the biogeochemical cycles. (Akala and Lal, 2001; Asensio et al., 2013; Santibañez et al., 2007; They also allow the soil to comply with its environmental and Vega et al., 2005; Zanuzzi et al., 2009). Another important problem productive functions, improving the previously existing environmental that affects these types of soils is the acid mine drainage, as it is a situation and constituting a process that simultaneously allows for the dangerous source of water contamination (Barrie and Hallberg, 2005; beneficial use of waste materials (Macías, 2004; Macías et al., 2007). Dold and Fontbote, 2001; Holmstrom and Ohlander, 2001). In order to The application of amendments allows for a large part of the carbon minimise all of these problems in mine soils, the use of amendments and nitrogen from this waste to become integrated in the soil. made of waste materials is a technique that can be carried out in situ Amendments made of waste are currently being used to recover mine soils in different parts of the world (Santibañez et al., 2007; Theodoratos et al., 2000; Zanuzzi et al., 2009; Zorzona et al., 2012). In ⁎ Corresponding author. Tel.: +34 986812630; fax: +34 986812556. this study, amendments were made using sludges from a bleach factory, E-mail address: [email protected] (R. Forján). solid urban waste, and another with sludges from a purification plant,

55 92 R. Forján et al. / Journal of Geochemical Exploration 147 (2014) 91–97 biomass and remnants from agri-food industries. These amendments the effect of adding each of the two amendments to the soil from the were produced by the company Tratamientos Ecológicos del Noroeste settling pool at the Touro mine, an experiment was designed using (T.E.N.) located in the same area where the soil used in this study was plant pots, which was carried out in a greenhouse for 3 months taken (Touro, Spain). The concentration of metals in assimilable form (Figs. 1–3). The greenhouse was maintained at an average temperature is important, as this is the way in which they can be absorbed and as- of 15 °C and a humidity of 70%. similated by organisms. As a result, when a metal that is toxic to some The plant pots were prepared with a soil-amendment ratio w:w of type of organism is in an assimilable form, it will be more dangerous 8:2 (S + A1 or S + A2). The amendments were deposited on the surface than when it is fixed in the soil (Adriano, 2001). The addition of organic of the soil without mixing them, in order to simulate the conditions amendments in degraded soils accelerates processes such as sorption, under which they are added in the field. Two leachates were collected precipitation and reactions for the formation of complexes that are pro- during the experiment, which were re-used to water their correspond- duced naturally in the soils to reduce the mobility and bioavailability of ing pot. The mixtures of S with A1 and A2 were named SA1 and SA2 re- metals (Bolan and Duraisamy, 2003; Hartley et al., 2004) thereby spectively. Unamended plant pots were used as a control, only avoiding their potentially toxic effects. containing soil (S). Twelve plant pots of each type were prepared in The novelty of this study lies in comparing the behaviour of two order to remove three of each kind in each time period (0, 1, 2 and amendments made of different types of waste on a mine soil. The 3 months). The different plant pots were watered three days a week main aim of this study was to evaluate the effect of two amendments with drop water. At the end of each time period, the soil samples were made of different wastes on the physical and chemical characteristics air dried and sieved through a 2 mm mesh in order to carry out the of a mine soil. analytical procedures. Particle size distribution was determined with the procedure of 2. Material and methods Kroetsch and Wang (2008). Soil reaction was determined with a pH electrode in 1:2.5 water to soil extracts. Electrical conductivity (EC) The sample zone is in the mine in Touro (north western Spain was determined according to Porta (1986). The method developed by (Lat/Lon (Datum ETRS89): 8° 20′ 12.06″ W42°52′ 46.18″ N). In order Mehra and Jackson (1960) was used to determine the free oxide con- to carry out the study, a soil was selected from the settling pool (S) at centrations. Aluminium, iron and manganese were determined in the the Touro mine, and two amendments made using waste materials extract by ICP-AES (Perkin-Elmer Optima 4300 DV). Both total and inor- (A1 and A2) provided by the company Tratamientos Ecológicos del ganic carbon (TC and IC) were determined in a module for solid analysis Noroeste (T.E.N.). (SSM-5000) coupled with a TOC analyser (Shimadzu TNM-1, Japan). The soil (S) was comprised of waste material resulting from the Soil organic carbon (SOC) contents were calculated from the difference flotation of sulphides during copper processing. The pool has been dry TC − IC. Dissolved organic carbon (DOC) was extracted with bidistilled for several years, and is considered to be soil according to the latest H2O according to Sáchez-Monedero et al. (1996) and the OC in the version of the FAO (2006). supernatant was determined with a TOC analyser (Shimadzu TNM-1, Amendment A1 consisted of a mixture of sludges from a bleach Japan). The different chemical organic matter fractions were separated plant, urban solid waste and material from the area around the mine following the method described in de Blas et al. (2010).Briefly, the (schist and natural soil). This amendment underwent a short maturity free organic matter (FOM) was removed from the soil samples by flota- process (less than one month). tion in 2 M H3PO4,centrifugationandfiltration of the supernatant. Fil- Amendment A2 consisted of sewage sludge from a purifying plant, ters with FOM were air dried and then weighed prior to analysing wood chips and remnants from agri-food industries. All of these compo- their carbon concentration in a solid module coupled with a TOC nents were mixed with soil from the area. This amendment was sieved analyser. The supernatant obtained after FOM extraction (soluble ex- after a long maturity period (more than one month). tract I) consisted of a phosphoric acid extracted FA fraction (FAP), The physical and chemical characteristics of the selected soil and which is not the FA fraction isolated through alkaline extraction of the both amendments are shown in Tables 1 and 2. In order to evaluate soil. The residue after FOM extraction (residue I) was subjected to two

Table 1 General characteristics, total nitrogen and carbon fractionation of the samples.

S A1 A2 SA1 SA2

pH (ms cm−1) 2.56e 8.51a 6.67b 3.63c 3.16d EC 2.29a 1.58b 0.77c 1.345b 1.04bc Sand % 83.25a 62.42d 27.15e 78.85d 71.82c Silt 14.91e 32.37b 64.26a 18.34d 24.78c Clay 1.83d 5.21b 8.57a 2.81 cd 3.41c Al oxides (mg kg−1) 852d 924c 4919a 866d 1665b Mn oxides 0.59d 357a 291b 71c 58c Fe oxides 22,903a 4470d 8263c 19,216b 19,975b TC (mg kg−1) 261c 16,729b 20,4890a 1079c 2894c OC 261e 13,442b 199,875a 797d 2618c IC u.l. 3287b 5014a 282c 276c

CFA 12.5d 42.6c 580a 13.2d 74.1b

CHA u.l. 66.1b 303a 10.6d 33.6c TN % 0.02d 0.18b 1.33a 0.09c 0.08c + −1 Na (cmol (+) kg ) 1.01d 2.90b 4.28a 0.82e 1.27c Mg2+ 0.2d 2.1b 5.2a 0.5c 1.8b K+ 0.08d 0.66c 5.71a 0.11d 1.07b Ca2+ 1.03c 2.92b 18.03a 1.40c 2.88b Al3+ 2.41a 0.0005c 0.001c 0.81b 0.84b H+ 6.83a 0.05c 0.06c 1.58b 2.04b

CECe 4.76c 8.57b 33.3a 3.66d 7.88b

CECt 11.5b 8.6d 33.3a 5.2e 9.9c For each row, different letters in different samples means signiﬁcantly differences (P b 0.05).

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Table 2 Pseudototal and DTPA-extractable concentrations of Cr, Cu, Ni, Pb and Zn in the soil (S), amendments (A1 and A2), and amended soils (SA1 and SA2); and Generic Reference Level established for Galician soils (GRL) of metals (Macías Vázquez and Calvo de Anta, 2009).

S A1 A2 SA1 SA2 GRL

Pseudototal Cr (mg kg−1) 236 ± 0.88a 61 ± 2.75c 73 ± 0.34c 201 ± 1.26b 204 ± 0.74b 80 Cu 1079 ± 43.5a 48 ± 2.07c 51 ± 1.29c 911 ± 35.1b 915 ± 34.4b 50 Ni 50 ± 0.85a 48 ± 2.07a 51 ± 1.29a 50 ± 0.83a 51 ± 1.19a 75 Pb 18 ± 1.11b 13 ± 0.44c 90 ± 1.99a 13 ± 0.25c 19 ± 0.85b 80 Zn 52 ± 1.66c 134 ± 4.07b 559 ± 19.4a 44 ± 0.74c 60 ± 0.95c 200 DTPA Cr u.l. 0.03 ± 0.00a 0.01 ± 0.00b u.l. u.l. extractable Cu 34.5 ± 0.28a 4.54 ± 0.28b 4.70 ± 0.42b 4.43 ± 0.07b 4.45 ± 0.04b Ni 1.48 ± 0.01a 0.45 ± 0.04d 0.61 ± 0.01d 1.14 ± 0.01b 0.87 ± 0.01c Pb u.l. 0.87 ± 0.00b 3.17 ± 0.00a u.l. 0.02 ± 0.00c Zn 0.64 ± 0.01d 3.59 ± 0.31b 32.37 ± 0.37a 0.66 ± 0.00d 1.92 ± 0.02c

For each row, different letters in different samples means signiﬁcantly differences (P b 0.05). u.l.: undetectable level. Detection limit of Cr, Cu, Ni, Pb and Zn are 0.01; 0.01; 0.01; 0.02 and 0.005 mg L-1.

successive extractions with 0.1 M Na4P2O7 prior to extracting CHA homogeneity, Dunnett's T3 test was performed. An independent t-test (humic acid carbon) and CFA (fulvic acid carbon), as NaOH can strongly was carried out in order to compare all of the soils when the data react with soil and consequently modify the results. After extractions were not parametric. A correlated bivariate analysis was also carried with Na4P2O7, successive extractions with 0.1 M NaOH (4–5 times) out with data from all of the soil samples. were carried out until the supernatant was transparent, indicating that all of the organic C should have been extracted. Supernatants 3. Results from Na4P2O7 and NaOH extractions from each soil sample were stored together as a homogeneous sample (soluble extract II). Supernatants 3.1. Physico-chemical characteristics of the mine soil (S), amendments were stored at 4 °C between extractions until the final analysis. TOC in (A1 and A2) and treated soils (SA1 and SA2) at the initial time the final sample was the extractable C (Cext) and was determined with a TOC analyser. Part of this combined sample was saved and H2SO4 The soil from the settling pond (S) had an acidic pH, while the two was added to allow the humic acids to precipitate. The supernatant amendments used (A1 and A2) had a significantly higher pH after this precipitation was analysed in the TOC equipment to determine (P b 0.05) (Table 1). The mine soil amended with these wastes (samples

CFA. The difference between Cext and CFA is the CHA. The residue after the SA1 and SA2 respectively) significantly increased their pH (Table 1). The last extraction with NaOH (residue II) corresponds to the humin frac- electrical conductivity (EC) was higher in S than in A1 and A2. The initial tion, which was dried and ground. Its organic C was determined in a samples of SA1 and SA2 had a lower EC than the unamended soil (S). solid module coupled with a TOC analyser. Soil S had a significantly higher percentage of sand than the amend- Total nitrogen (TN) was determined by the Kjeldahl method modi- ments and treated soils. A1 and A2 had a higher percentage of clay and fied by Bremmer and Mulvaney (1982) using the equipment Tecator silt in comparison with S. Silt content in SA1 and SA2 was significantly Kjeltec System 1026, combined with a digestion unit (DK 20 Heatinh higher than in S, and in the case of SA2 the percentage of clay also in- Digester). creased (P b 0.05) (Table 1). Exchangeable cations (Ca2+,K+,Mg2+,Na+ and Al3+)wereex- Soil S had higher iron oxide content than the amendments (Table 1). tracted with 0.1 M BaCl2 (Hendershot and Duquette, 1986) and element Samples A1 and A2 had high concentration of manganese oxides in concentrations were determined by ICP-OES (Perkin-Elmer Optima comparison to S (Table 1). The amendments had a significantly higher 4300 DV). Exchangeable hydrogen (H+) was extracted with 1 M KCl concentration of total organic carbon (TOC) and inorganic carbon (IC) and its concentration was determined by titration with NaOH than the soil from the settling pond. SA1 and SA2 also had significantly (0.01 M) and titrated with phenolphthalein (1%) (Thomas, 1982). higher concentration than S, indicating how effective these amend- Effective cation exchange capacity (CECe) was calculated with the ments can be in contributing carbon (P b 0.05) (Table 1). Samples A1 total cation concentration. A series of critical values were assigned to and A2 had significantly higher concentration of fulvic acid carbon each of the chemical parameters, based on the model of the SFCC (Soil (CFA) and humic acid carbon (CHA) in comparison to S (P b 0.05) Fertility Capability Classification) proposed by Buol et al. (1975) and (Table 1). Therefore, both treatments increased the concentration of adapted by Macías and Calvo (1983), Calvo de Anta and Macías these humified forms of C in the soil from the settling pool. (1987), Calvo et al. (1987). These were used to evaluate the limiting The percentage of total nitrogen was higher in A1, A2, SA1 and SA2 in factors for plant production. comparison to S (P b 0.05) (Table 1). The metals in assimilable form were extracted using a solution of The Al3+ and H+ content in S was significantly higher than that in DTPA (Lindsay and Norvell, 1978). The pseudototal metal concentration the amendments and treatments (P b 0.05) (Table 1). The amendments was determined using acid digestion with aqua regia in a microwave had higher concentrations of Ca2+,K+,Mg2+ and Na+ and a higher oven (Milestone ETHOS 1). The concentration of the metals in the solu- effective cation exchange capacity than S. The treated soil A2, as well tions was measured using an ICP-OES device. The certified reference as increasing the CECe, also increased the CECt (P b 0.05). Both treat- material CRM026 was analysed in parallel with samples to check the ef- ments had a significantly higher content of Mg2+ in comparison to fectiveness and precision of the extraction analysis. The pseudototal soil S. SA2 also had a significantly higher content of Ca2+and K+ concentrations of the metals being studied were compared with the (P b 0.05) (Table 1). maximum values established in the Generic Reference Levels for Galicia Soil S had a pseudo-total content of Cr and Cu above the Generic Ref- (GRL) (Macías Vázquez and Calvo de Anta, 2009). erence Level (GRL), which was also significantly higher than the amend- All of the analytical determinations were performed in triplicate. The ments and treated soils (P b 0.05) (Table 2). Amendment A1 was not data obtained were statistically treated with the programme SPSS ver- contaminated by any of the metals that were analysed. The soil sion 19.0 for Windows. Analysis of variance (ANOVA) and test of homo- amended with A1 (SA1), despite having a reduction of the pseudototal geneity of variance were carried out. In case of homogeneity, a post-hoc concentrations of Cr and Cu by 23% and 15% respectively with regard least significant difference (LSD) test was carried out. If there was no to S, was also contaminated by Cr and Cu. Although amendment A2

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Fig. 1. Soil pH, electrical conductivity (EC), exchangeable cations and total and effective cation exchange capacity (CECt and CECe) in the mine soil (S) and the treated (SA1 and SA2) at 1 and 3 months. Different letters in different bars each time means differences (P b 0.05). had a pseudototal concentration of Pb and Zn above the GRL and sig- 3.2. Evolution of the physical–chemical characteristics in the amended and nificantly higher than soil S (Table 2), SA2 was not contaminated by unamended soil over the 3-month period these metals and their concentrations were significantly the same as in S (P b 0.05). However, SA2 was contaminated by Cr and Cu. The The amended soils (SA1 and SA2) had significantly higher pH than concentrations of DTPA-extractable Cu and Ni were significantly the untreated soil from the settling pool (S) both one month and three higher in S in comparison with the amendments and treatments months after adding the amendments (P b 0.05) (Fig. 1). (P b 0.05) (Table 2). The amendments had higher concentration of Soil S had a higher electrical conductivity (EC) than SA1 and SA2 in DTPA-extractable Cr, Pb and Zn than S, in particular the concentra- the first month. However, these differences disappeared two months tions of Pb and Zn in A2, whose values were respectively 3 and 30 later (P b 0.05) (Fig. 1). times higher than in S. When applied to S, amendment A2 led to Soil S had a concentration of acid cations (Al3+ and H+)inthefirst SA2 having a higher DTPA-extractable content of Pb and Zn than S month that was significantly higher (P b 0.05) than that of SA1 and (P b 0.05) (Table 1). SA2, which continued over the three-month period (Fig. 1). The basic

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Fig. 2. Concentrations of organic carbon (OC), inorganic carbon (IC), fulvic acid carbon (CFA), humic acid carbon (CHA) and percentage of total nitrogen (TN) in soil (S) and amended soils (SA1 and SA2) at 1 and 3 months. Different letters in different bars each time means differences (P b 0.05). u.l.: undetectable level.

Fig. 3. DTPA-extractable concentrations of Cu, Ni, Pb and Zn in the soil (S) and amended soils (SA1 and SA2) at 1 month and 3 months. Different letters in different bars each time means differences (P b 0.05). u.l.: undetectable level.

59 96 R. Forján et al. / Journal of Geochemical Exploration 147 (2014) 91–97 cations predominated in SA1 and SA2. In soil SA2, in comparison to S, The application of amendments made from solid urban waste and there was also a higher CECe and CECt (P b 0.05) (Fig. 1). sludge from the purification plant sometimes results in an additional With regard to the different carbon fractions, it is important to note contribution of metals to the soil being treated (Smith, 2009; Weber the high content of TOC, CFA and CHA in SA2 (Fig. 2). Both treatments et al., 2007). In the case of the amendments used in this study, although presented significantly higher concentrations of TOC, IC, CFA and CHA there was no significant increase in the pseudototal concentration of in comparison to S in each of the months of the experiment (P b 0.05) any of the metals, the concentrations of Cu, Pb and Zn in assimilable (Fig. 2). form did increase. The concentration of TN was significantly higher in SA2 than in S in The increase in the concentration of assimilable Cu in the soil all of the months, but not in SA1 (P b 0.05) (Fig. 2). amended with A2 in the last month is probably due to the decrease in Soil S had a concentration of DTPA-extractable Ni that was signifi- the pH over the three month period. In fact, a negative correlation was cantly higher than its initial concentration (Table 2) after one month obtained between the pH and the DTPA-extractable Cu content in SA2 of the experiment. These differences disappeared in the third month, (r = −0.93, P b 0.01). The DTPA-extractable Zn content also increased and even equalled the concentrations of the treated soils (P b 0.05) over time in the two amended soils, and a negative correlation was also (Fig. 3). SA1 had a DTPA-extractable Zn concentration that was higher obtained between the DTPA-extractable Zn and the pH in each of them than that of S both in the first and third month, and of Pb in the third (r = −0.85, P b 0.05 in SA1 and r = −0.93, P b 0.01 in SA2). Therefore, it month (Fig. 3). SA2 had significantly higher concentrations of DTPA- would be important to keep the pH in the mine soil as basic as possible extractable Cu, Zn and Pb than those in S in both the first and third in order to overcome the problem of the high metal concentrations, and months (P b 0.05) (Fig. 3). at the same time that the contribution of carbon and nitrogen from the amendments can be used to the greatest possible extent by soil 4. Discussion organisms. The DTPA-extractable contents of Ni and Pb were also negatively The significantly higher pH level in the two treated soils (SA1 and correlated with the pH, with a Pearson's correlation coefficient of SA2) with respect to the untreated soil (S) was due to the contribution more than 0.86 in all cases (P b 0.05). This makes it possible to deduce of basic cations from the amendments used (A1 and A2). The EC in- that the amendments made using solid urban waste and sludge from crease due to the used wasted, because the watering and degrading of a purification plant contribute these metallic elements in both a total organic material cause solubilization of present salts. The amended form and DTPA-extractable form (Brown et al., 2003; Madrid et al., soil is not considered degraded by the EC because the value does not ex- 2007; Paradelo et al., 2011; Pichtel and Anderson, 1997; Zorzona et al., ceed 6 (ms cm−1)(FAO, 1998). This increase in the pH is very important 2012). The concentration of Cu is therefore due to both the waste for the mine soil, because it entails reducing the mobility of the metals used and the base material for soil S. In order to avoid the negative effect and making more nutrients available for the soil organisms. Other of the amendments, which can increase metal concentrations in soils; it authors have previously stated that the types of waste used in this would be necessary to use a higher proportion of amendment or re- study (solid urban waste, ash from a paper company, remnants from amendment to extend in time the positive effects. agri-food industries and sludge from a purification plant) have high concentrations of basic cations (Amir et al., 2005; Weber et al., 2007). 5. Conclusions Apart from raising the acidic pH of the mine soil, the increase in the concentration of these cations is important as they serve as nutrients for liv- The application of different types of waste materials (sludge from a ing organisms in the soil. The concentration of basic cations also entailed bleach factory, solid urban waste, sludge from a purification plant, an increase of the CECe (P b 0.01), which is associated with processes wood chips and remnants from agri-food industries) to the soil of the for the sorption and immobilisation of metals in the soil (Kumpiene settling pond significantly improved its conditions, as they increased et al., 2008). These cations are also important because together with its pH, CEC, C and N. Amending degraded soils with these wastes may the OC, the biota, clay and carbonates, they form stable aggregates in entail other unwanted effects such as increasing the concentration of the soil that help with its correct structuring (Bromick and Lal, 2005). metals from the waste products used. The results obtained show that In order to maintain high soil pH for longe time it would be used higher the sludge from the purification plant was the waste that resulted in proportion of amendemt or re-amend. the worst consequences in this respect. After obtaining these results, it The untreated soil (S) was poor in OC and IC. The contents of these is necessary to evaluate if the rapid rise in the levels of C, N, pH and nu- types of C increased considerably in SA1 and SA2. This contributed trients compensates for the increase in the DTPA-extractable content of carbon came from the waste in A1, such as the solid urban waste certain metals; or if otherwise it would be better to increase these con- (Illera et al., 2000; Paradelo et al., 2011; Pichtel and Anderson, 1997) tents more slowly, which does not increase the content of DTPA- and from A2, such as the sludge from the purification plant and rem- extractable metals as was the case with SA1. For all of these reasons, it nants from the agri-food industries (Canet et al., 2007; Nicholson is important to evaluate the use of the soil when applying these amend- et al., 1996; Pichtel and Anderson, 1997; Yujun et al., 2011). The carbon ments. In the case of mine soils, all of the waste materials that were contribution is important for the recovery of degraded soils, as the in- studied would be valid, as the pool soil is contaminated by metals and crease in organic carbon in the soil increases its possibility to retain cannot be used for agricultural purposes. In the case of soil used for the water that is available, increases the concentration of nutrients in non-industrial purposes or purely for environmental recovery, amend- bioavailable form, and improves the structure of the soil and other ment with sludges from purification plants would not be a valid physical properties (Lal, 2006). The increase in the carbon contents solution. from fulvic and humic acids (CFA and CHA) in both treatments is impor- fi tant, as these types of C, apart from bene ting the structure and func- References tions of the soil, also interact with metal ions to reduce the toxicity, and are important in plant growth (Amir et al., 2005; Jindo et al., Adriano, D.C., 2001. Trace elements in terrestrial environments. Biogeochemistry, Bio- 2012; Muscolo et al., 2013). In order to maintain the stability of soil availability and Risk of Metals. Springer, New York. E.E.U.U. Akala, V.A., Lal, R., 2001. Soil organic carbon pools and sequestration rates in reclaimed carbon over time it would be necessary to use a proportion higher minesoils in Ohio. J. Environ. Qual. 30, 2098–2104. than 8:2 for soil:amendment. If this experiment would be carried out Amir, S., Hafidi, M., Merlina, G., Revel, J., 2005. Sequential extraction of heavy metals on the field, it would be necessary to know its evolution and re- during composting of sewage sludge. Chemosphere 59, 801–810. fi Asensio, V., Vega, F.A., Sing, B.R., Covelo, F., 2013. Effects of tree vegetation and waste amend to stabilize. The signi cant increase in the amount of nitrogen amendments on the fractionation of Cr, Cu, Ni, Pb and Zn in polluted mine soils. in the case of SA2 was due to the waste used to make amendment A2. Sci. Total Environ. 443, 446–453.

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2.2 Contribution of waste and biochar amendment to the sorption of metals in a copper mine tailing

Catena 137 (2016) 120–125

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Contribution of waste and biochar amendment to the sorption of metals in a copper mine tailing

R. Forján a,⁎, V. Asensio b, A. Rodríguez-Vila a,E.F.Coveloa a Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, As Lagoas-Marcosende, 36310 Vigo, Pontevedra, Spain b Department of Plant Nutrition, CENA, University of São Paulo (CENA-USP), Av. Centenário 303, 13400-970, Piracicaba, SP, Brazil article info abstract

Article history: One technique applied to restore degraded or contaminated soils is to use amendments made of different types of Received 3 December 2014 waste materials, which in turn may contain metals such as Cu, Pb and Zn. For this reason it is important to deter- Received in revised form 2 September 2015 mine the capacity of the soil to retain these materials, and to compare the sorption capacity between an amended Accepted 18 September 2015 soil and another unamended soil. The aim of this study was to determine the chemical behaviour of these metals Available online 29 September 2015 in the soil after applying the amendment, and how it affected the soil's sorption capacity. Another aim was Keywords: to study the contribution of contaminating elements from the amendment itself. The amendments used in this Wastes study were a mixture made of waste material (sewage sludges, sludges from an aluminium plant, ash, food indus- Mine soil try wastes, and sands from a wastewater treatment plant) and biochar (biomass of Acacia dealbata)(97%:3%)in Metal different soil/amendment proportions. The soil was from a mine tailing. The mine tailings were amended with the Soil remediation mixture of waste and biochar which had a higher sorption capacity than the soil from the pond. The samples with Biochar amendment had a greater afﬁnity for Cu, Pb and Zn than the mine soil. The results obtained show that adding a Sorption mixture made of waste and biochar favours the retention of Cu, Pb and Zn in mine tailing from metal mines. © 2015 Elsevier B.V. All rights reserved.

1. Introduction leads to a descent in the assimilable metal content. Organic matter also adds essential nutrients that improve the fertility level of the soils, and Mine soils are highly degraded at both physical and chemical levels, helps to improve certain physical–chemical properties of the soil (Illera which prevents them from carrying out their functions and plants et al., 2000). Sometimes, the waste material used as an amendment con- growing on their surface. Mine soils are a source of pollution that affects tains contaminants such as metals or organic compounds, which means the area where the mine operates and the adjacent areas. This is due to that many of them cannot be used on any type of soil. It is also necessary the contamination of underground waters as a result of acid runoff from to control the proportion of amendment/soil used, in order to ensure a the mine, or atmospheric pollution caused by fine particles that are car- balance between their potentially beneficial qualities and possible prob- ried off into the air. One of the main impacts that mining has on soils is lems of contamination (Paradelo et al., 2011). Adding organic matter to their high concentration of metals, acidity and low organic matter con- these types of soils leads to a descent in the assimilable metal content, tent (Akala and Lal, 2001; Vega et al., 2005; Santibañez et al., 2007; as this is capable of forming strong bonds with metals and therefore re- Zanuzzi et al., 2009; Asensio et al., 2013). It is important to understand tain them firmly in the soil (Kabata-Pendias, 2001). Different studies how these metallic elements behave to their concentration and the ease have shown that adding amendments made of waste materials reduces with which they migrate through these types of soils. the assimilable metal content in the soil (Brown et al., 2003; One of the most recently used techniques to recover mine soils in- Natal-da-Luz et al., 2012). The increased content of organic matter pro- volves amendments made in situ with different types of waste material vided by the residue causes an increase in the sorption capacity of (such as municipal solid wastes ‘(MSW)’,sewagesludgeorwastefrom heavy metals in soil (Kocasoy and Güvener, 2009). These studies were the food industry). Using these types of amendments has a double bene- based on understanding how the available amount of metals varied in fit. Firstly, it returns the components of these wastes to the biogeochem- the soil, but not on how these metals behaved in the soil once the amend- ical cycle, and secondly, it adds organic matter to the soil, increasing ments had been applied. the pH and immobilizing metals (Theodoratos et al., 2000; Paradelo Another option, despite still being costly, is to use biochar in combi- et al., 2011). The contribution of organic matter is important, as previous nation with these amendments to recover degraded soils. Biochar is ob- studies (Karami et al., 2011) have shown that increasing it in the soil tained by biomass pyrolysis (using both plant and animal waste) using substoichiometric combustion or without oxygen in pyrolytic ovens. ⁎ Corresponding author. The biochar, in the same way as the amendments made using waste E-mail address: [email protected] (R. Forján). material, provides organic matter, especially carbon and nitrogen.

65 R. Forján et al. / Catena 137 (2016) 120–125 121

However, unlike the amendments, the waste used to make biochar usual- and Gomes et al. (2001)) modified by Harter and Naidu (2001).We ly contains very few or no trace elements, such as forestry biomass. They used single-metal solutions of Cu2+,Pb2+ and Zn2+ nitrates (0.03, −1 also have the added benefit that the C and N they provide are much more 0.05, 0.08, 0.1 and 0.5 mmol L ) containing 0.01 M NaNO3 as back- recalcitrant. Different studies (Beesley et al., 2010; Beesley and Marmiroli, ground electrolyte (Vega et al., 2009). Soil samples (1.5 g) were shaken 2011; Park et al., 2011) have shown that biochar can reduce available with 25 mL of “sorption solutions” in polyethylene tubes in a rotatory contents of metals such as Cu, Pb and Zn, and increase the soil pH. The bio- shaker for 24 h at 25 °C. The samples were then centrifuged 10 min char used in this study was made using Acacia dealbata Link. This is a at 3000 rpm and the supernatant was filtered through Whatman 42 shrub from the legume family (Fabaceae) that is considered an invasive paper (pore size 0.45 μm). Supernatants were analysed by ICP-AES species in the Iberian Peninsula. Using biochar made of A. dealbata as (Perkin-Elmer Optima 4300 DV) to determine the Cu, Pb and Zn concen- an amendment would therefore serve two functions — recovering the trations. The concentration of each metal that had been sorbed by each soil and using the waste matter from a type of invasive plant. The biochar soil sample was calculated from the difference between its concentra- increases the sorption capacity of heavy metals in soil; this increase is due tion in solution before the addition of this soil and after equilibration to the high pH, organic matter, and large surface area (Beesley et al., 2010, (shaking) with soil. The sorption isotherms were constructed for each Beesley and Marmiroli, 2011; Uchimiya et al., 2011). metal by plotting the sorbed metal concentration of the soil sample An understanding of metal sorption processes can help in providing (μmol per g of dry soil) against the concentration of the metal in solu- very valuable information about the effect of amendments once they tion at equilibrium (μmol L−1). To take exchange into account the back- have been applied to the soil. Therefore, the aim of this study was to com- ground electrolyte, the concentrations of the studied metals were pare the sorption capacity of Cu, Pb and Zn in a copper mine soil after ap- corrected by subtracting the values determined in an additional exper- plying a combined amendment of waste and biochar, and to understand iment, in which the sorption solution contained only 0.01 M NaNO3.All the behaviour of the metals included in the amendments. Copper, lead of the sorption experiments were performed in triplicate. and zinc are usually found in high concentrations in the soils from the For each metal sorption isotherms were constructed by plotting the mine in the study, and in the waste materials used as amendments. sorbed metal concentration (μmol per g of dry soil sample) against the concentration of the metal in solution at equilibrium (μmol L−1). The 2. Material and methods obtained isotherms were compared with to the models of Langmuir (1) and Freundlich (2) and with the types of curve described by Giles 2.1. Study area and amendments et al. (1974). The overall capacity of the soils to sorb Cu, Pb or Zn was evaluated as the slope Kr (3) according to Vega et al. (2008). The sample zone is in the mine in Touro (Northwest Spain; Lat/Lon (Datum ETRS89): 8° 20′ 12.06″ W42°52′ 46.18″ N). The climate in (1) Langmuir model: this zone is Atlantic (oceanic) with precipitation reaching 1886 mm per year (with an average of 157 mm per month) and a mean daily tem- x/m_CK β /1+K C. perature of 12.6 °C. The average of relative humidity is 77% (AEMET L L L 2014). For this study, a mine tailing was chosen from the mine's settling xquantityofmaterialsorbed(μmol). pond (S). The amendment made of wastes was provided by the compa- m quantity of sorbent (g). ny Tratamiento Ecológicos del Noroeste (T.E.N.). This amendment C Solution concentration equilibrium (μmol L−1). was made of a mixture of sewage sludges, sludges from an aluminium K Langmuir constant (L μmol L−1). plant, ash, food industry wastes, and sands from a wastewater treat- L β high sorption capacity (μmol g−1) ment plant (T). The biochar (B) was made of biomass from areas in- L vaded by A. dealbata in the north of Portugal, and was obtained by (2) Freundlich model: pyrolysis, without oxygen, at 450 °C of temperature during 8 h.

Log x/m=1/n logC+ log KF. 2.2. Experiment design and soil chemical analyses xquantityofmaterialsorbed(μmol). This mine tailing was treated with a mixed amendment consisting m quantity of sorbent (g). of a mixture of different wastes (T) combined with biochar (B). The C Solution concentration equilibrium (μmol L−1). amendment (TB) and the mine tailing (S) were mixed (STB) at 20, 40, −1 KF Freundlich constant (L g ). 60% and then placed in glass vessels. The mixture of the amendment ndimensionless.nN 1, sorption is favourable made of wastes and biochar was called TB. The amendment TB and the mine tailing S at 100% were also placed in glass vials as control sam- (3) Kr ples. The amendment TB was made with 97% wastes and 3% biochar. All mixtures were made w/w. The mixtures and controls were incubated in It is a line of type y = ax darkness to ﬁeld capacity for one month. Soil pH was determined with a pH electrode in 1:2.5 to soil extracts. y quantity of sorbent (μmol g−1). The phytoavailable content of Cu, Pb and Zn was extracted with 0.01 M x amount of added metal or metals per gramme of soil (de-

CaCl2 in soil solution (Houba et al., 2000). Quasitotal metal contents duced from the concentration of the solution with which were extracted with aqua regia by acid digestion in a microwave oven was treated) versus sorbed amount of each of the metals (Milestone ETHOS 1, Italy) and analysed in an ICP-OES. Soil total carbon (μmol g−1).

(TC) was determined in a solid module (Shimadzu SSM-5000, Japan) aistheKr parameter. It is the slope of the line. It is a dimension- coupled with a TOC analyser (Shimadzu TNM-1, Japan). These analyses less parameter. If a ≈ 1highafﬁnity for the metal. were carried out with soil S, the mixtures and the positive control TB100%. 2.4. Statistical analyses 2.3. Sorption experiment and construction of isotherms All of the analytical determinations were performed in triplicate. The To evaluate the sorption capacity of Cu, Pb and Zn by the mine tailing data obtained were statistically treated with the program SPSS version and the different mixtures, data for isotherm construction were ob- 19.0 for Windows. Analysis of variance (ANOVA) and test of homogene- tained in batch experiments using the method of Alberti et al. (1997) ity of variance were carried out. In case of homogeneity, a post-hoc least

66 122 R. Forján et al. / Catena 137 (2016) 120–125 signiﬁcant difference (LSD) test was carried out. If there was no homogeneity, Dunnett's T3 test was performed. An independent t-test was carried out in order to compare all of the soils when the data were not parametric. A correlated bivariate analysis was also carried out with data from all of the soil samples.

3. Results

3.1. Characteristics of the mine tailing (S), amendment (T) and biochar (B)

The mine tailing (S) had an acidic pH, while the amendment and the biochar used (T and B, respectively) had a signiﬁcantly higher pH than the untreated soil (Table 1). The biochar and the amendment also had a signiﬁcantly higher concentration of carbon (C) than the mine soil.

The highest concentration of both quasitotal and CaCl2-extractable Cu was observed in the soil, and the amendment and biochar had concen- Fig. 1. pH values of the different treatments. trations signiﬁcantly lower. The highest concentration of quasitotal

CaCl2-extractable both Pb and Zn were observed in the amendment and biochar) and the mixtures STB60% and STB40% have a high affinity (Table 1). for this metal, indicated by the initial slope of the isotherms. On the contrary, the sorption isotherm for Cu by S shows that virtually all of 3.2. Characteristics of the mine tailing (S), mine tailing amended with the metal remains in the equilibrium solution without being absorbed amendment mixtures (STB20, STB40%, STB60%) and biochar amendment by the mine tailing. As shown in Fig. 3, the sorption isotherms for Pb (TB100%) have an initially steep slope for TB100%, STB60% and STB40%, which have the greatest affinity for this metal. The slope of the sorption iso- All of the samples that contained amendment + biochar (20, 40, 60 therm for STB20% was less steep, although it did have a higher affinity and 100% proportion of amendment + biochar:soil) had a significantly for Pb than S. Mine tailing S had a sorption isotherm with an initially higher pH than soil from the mine tailing or the biochar (Fig. 1). We can flat slope, but which did increase slightly at the end (Fig. 3). TB100%, see that the higher the proportion of amendment, the higher the pH of STB60% and STB40% have the greatest affinity for Zn according to their the sample. isotherms. As can be seen in Fig. 3, in the case of Zn the isotherm

Fig. 2 shows the quasitotal and CaCl2-extractable concentrations of for the mixture STB20% practically all of the metal remains in the equi- Cu, Pb and Zn 2. As may be seen, there were no significant differences librium solution, on the contrary to the situation with this same mixture between the quasitotal Cu content in the unamended mine tailing for Cu and Pb. In this case, neither does mine tailing S sorb this metal. (S) and the soil with the different proportions of amendment (ST20%, Sorption capacity of the mine tailing without amendment is very low STB40%, STB60%). However, there were differences with the positive control (TB100%) which had a lower quasitotal Cu content than S and ST20%, STB40% and STB60% (Fig. 2). The quasitotal contents of Pb and Zn revealed a tendency to increase in line with the percentage of amendment applied to the mine tailing (Fig. 2). The CaCl2-extractable Cu content revealed significant differences, with the highest content in the untreated mine tailing S and the mixture STB20% (Fig. 2). In the mixtures STB20%, STB40% and STB60% a decreasing trend in the CaCl2- extractable Cu content was observed as the amount of amendment applied increased (Fig. 2). In the case of the CaCl2-extractable Pb and Zn content, the trend was the opposite to that seen for Cu, with STB20%, 40% and 60% having a significantly higher content (Fig. 2). This trend was much more pronounced in the case of Zn, whose contents were much higher than for Pb.

3.3. Sorption isotherms of Cu, Pb and Zn

Fig. 3 shows the individual sorption isotherms for Cu, Pb and Zn. As can be seen, in the case of Cu, the positive control TB100% (amendment

Table 1 Characteristics of the mine tailing (S), amendment (T) and biochar (B).

STB

pH 2.62 ± 0.01c 5.54 ± 0.1b 9.44 ± 0.1a C(gkg−1) b5·10−5 254 ± b 672 ± 5.5a Cu Quasitotal 636 ± 50a 290 ± 57b 27.7 ± 0.9c Pb(mg kg−1) 8.21 ± 0.8b 90.1 ± 22a u.d Zn 63.7 ± 4.1b 771 ± 33a 64.5 ± 0.7b

Cu CaCl2-extractable 138 ± 13a 1.83 ± 0.1b u.d Pb(mg kg−1) 0.28 ± 0.1a 0.34 ± 0,1a u.d Zn 2.41 ± 0.01b 164 ± 5.1a 1.31 ± 0.1c

For each row, different letters in different samples mean signiﬁcant differences (P b 0.05). Limit of detection = 5 · 10−5. u.d = under level detection. Fig. 2. Quasitotal and CaCl2-extractable Cu, Pb and Zn concentrations.

67 R. Forján et al. / Catena 137 (2016) 120–125 123

Fig. 4. Distribution coefﬁcient (Kr) for the individual sorption of Cu, Pb and Zn.

control sample (S) did have a signiﬁcantly lower sorption capacity for

Pb than the amended samples (Fig. 4). The Kr obtained for Zn indicated that soil S has a zero sorption capacity for Zn. All of the mixtures – STB20%, STB40% and STB60% – and the control sample TB100% had a signiﬁcantly higher sorption capacity for Zn than S, with STB60% and TB100% having the highest sorption capacity (Fig. 4).

3.5. Selectivity sequences for Cu, Pb and Zn

Table 2 shows a comparison of the sorption preferences of mine tailing S, the mixtures (STB20%, STB40%, STB60%) and the positive con-

trol (TB100%) for Cu, Pb and Zn based on the Kr calculated. In all of the samples that were studied, the metal that was preferably sorbed was Pb. The Cu and Zn varied their position in the selectivity sequences. The mine tailing, STB40% and STB60% followed the sequence Pb N Cu N Zn, while the sequence for the mixture STB20% and positive control was Pb N Zn N Cu.

4. Discussion

Fig. 3. Sorption isotherms of Cu, Pb and Zn. 4.1. Characteristics of the mine tailing (S), mine spoil material amended with amendment mixtures (STB20, STB40%, STB60%) and biochar amendment (TB100%) due to its sandy loam texture, low pH and low organic matter content (Weng et al., 2001).As regards the Langmuir and Freundlich adjust- The pH value of the mine tailing (S) amended with different mix- ments, not all of the isotherms fitted to them. In the case of the sorption tures of waste material and biochar (STB20%, STB40%, STB60%) con- isotherms of Cu, these fitted the Freundlich model, except for S, with the stantly increased as the proportion of amendment applied increased. r2 for STB20%, STB40%, STB60% and TB100% being 0.96; 0.97; 0.96 and This increase in the pH value is due to the waste matter used to make 0.98 respectively. Mine tailing S fitted the Langmuir model with an r2 the amendment, such as solid urban waste, ash from a paper factory, of 1. Only the sorption isotherms of Pb for S, STB20% and STB60% fitted agri-food waste, purification plant sludge and biochar (Canet et al., the Freundlich model, with r2 of 0.81; 0.96; and 0.8 respectively. The 2007; Hackett et al., 1999; Nicholson et al., 1996; Tejada et al., 2010). sorption isotherms for Pb both for STB40% and for TB100% did not This increase in the pH is important because it reduces the mobility of fit either the Langmuir model or the Freundlich model. The sorption the metals in the soil, and also means that there are more nutrients in isotherms for Zn fitted the Freundlich model, except for STB40%, with an available form. 2 r for S, STB20%, STB60% and TB100% of 0.96; 0.97; 0.98 and 0.97 respec- The CaCl2-extractable concentrations of Cu, Pb and Zn varied greatly tively. The sorption isotherm of Zn for STB40% did not fit either the depending on the metal and the percentage of the mixture applied

Langmuir model or the Freundlich model. to the mine tailing. For this reason, the percentage of the CaCl2- extractable/Quasitotal ratio was calculated. In the case of the percentage

3.4. Estimation of the sorption capacity using the distribution coefficient Kr of CaCl2-extractable Cu and Pb content with respect to the quasitotal, this decreases if we compare mine tailing with the mixtures. This de- Having observed that not all of the isotherms were a close fitto crease varies for Cu from a value of 21.7% for S, to 6.2% for STB40% and the Langmuir and Freundlich models, the distribution coefficient Kr was calculated in order to estimate the sorption capacity. As shown in Table 2 Comparison of the sorption capacity of the mine tailing with the Fig. 4,theKr obtained for Cu showed that mine tailing S has a zero sorption capacity for this metal. In turn, the amendment (TB100%) had the mixtures and amendment. same Cu sorption capacity as STB60% and STB40% with Kr of 0.9776, Mine tailing Selectivity sequence 0.9805 and 0.9745 respectively. These three mixtures have more sorp- SPbN Cu = Zn tion capacity for this metal than STB20% (Fig. 4). The Kr obtained for Pb STB20% Pb = Zn N Cu indicates that there are no significant differences between the sorption STB40% Pb = Cu N Zn capacity for this metal amongst all of the samples that contain amend- STB60% Pb N Cu N Zn TB100% Pb N Zn N Cu ments, including the pure amendment (TB100%). The unamended

68 124 R. Forján et al. / Catena 137 (2016) 120–125

0.5% for STB60%. In the case of Pb, S has an assimilable percentage with reason why the sorption capacity increased in S is possibly due to the in- respect to the total of 3.4% and dropping to 1.3% in mixture STB60%. This crease in the amount of organic matter, once the soil had been amended decrease in the percentage of the available content in comparison to in its different proportions (STB20%, STB40%, STB60%), as it has been the quasitotal is due to the increase in the organic matter content and demonstrated that soils with a higher organic matter content have a in the pH, that is, the metal was fixed to the mine tailing due to the effect higher capacity to retain metals (Covelo et al., 2007; Temminghoff of the amendment (Canet et al., 2007; Hackett et al., 1999; Park et al., et al., 1997). One of the reasons for this increased amount of organic 2011; Nicholson et al., 1996; Tejada et al., 2010). pH plays a crucial matter could be due to the waste used to make amendment T: solid role in metal solubility; it is well known that a high pH favours metal urban waste, ash from the paper industry, waste from agri-food indus- sorption in soils. It has also been demonstrated that soils with a higher tries, and sludges from purification plants (Nicholson et al., 1996; organic matter content have a higher capacity to retain metals (Covelo Canet et al., 2007; Hackett et al., 1999; Tejada et al., 2010). et al., 2007; Park et al., 2011; Temminghoff et al., 1997; Weng et al., The contribution of metallic elements from the amendment applied 2001). Biochar causes an increase in the metal sorption capacity because would be mitigated by its high sorption capacity. For example, in mine it has a high pH, organic matter content and large surface area (Beesley tailing S, the percentage of CaCl2-extractable Cu in comparison to the et al., 2010). quasitotal Cu content is 21%, and for Pb is 3.4%. In sample STB60% the percentage is 0.53% for Cu and 1.33 for Pb. This shows that despite 4.2. Sorption capacity of mine tailing for Ni, Pb and Zn contributing metallic elements, the amendment would be beneficial, as it has a high sorption capacity for metals, contributes organic matter The isotherms for Cu resemble H-type curves, or with a very high af- and increases the pH of S. finity (Giles et al., 1974)(Fig. 3). The isotherms for Pb, both for TB100% Recovering mine tailing by applying this organic amendment, and STB60% resemble an H-type curve, while for STB40% they resemble consisting of a combination of a classic amendment using waste mate- an L-type curve. The isotherms for Zn both for TB100% and STB60% re- rials (solid urban waste, ashes from a paper factory, agri-food waste semble an H-type curve, while the isotherm for Zn for STB40% resem- and sludges from a purification plant), together with biochar, signifi- bles an L-type curve, as with Pb (Fig. 3). cantly increases the sorption capacity of Cu, Pb and Zn in all of the pro- The mine tailing without any type of treatment (S) has a zero sorp- portions used (20%, 40% and 60%), being most effective in those in tion capacity for Cu and Zn; in the case of lead sorption peak it is possi- which 40% and 60% were applied (Fig. 4). This is because the higher bly because lead is a metal that has more affinity for soil binding sites, the proportion of amendment in the soil, the greater the contribution and also for a possible minimum and temporary increase in organic of carbon and increase in the pH. matter (Fig. 4). The sorption isotherms for these three metals indicated that all of their contents remained in the equilibrium solution, indi- 4.3. Selectivity sequences for Cu, Pb and Zn cating a non-existent sorption capacity for these metals (Fig. 3). This may be due to the low organic matter content and highly acidic pH of The selectivity sequences of Cu, Pb and Zn revealed different be- this soil. This low sorption capacity indicates that the metals studied haviours between STB20%, STB40%, STB60% and TB100%, without it would be in an available form in mine tailing, which would favour dif- being possible to define a pattern. This is mainly due to the heterogene- ferent problems such as acid runoff from the mine or increased contam- ity of the amendments made using wastes. This heterogeneity is due ination of the surrounding areas. to the fact that the metals are not distributed uniformly through the The zero sorption capacity of S increased significantly once it had amendment. It is important to note the high sorption capacity of the been amended with the different mixtures used in this study – STB20%, amendments for these metals, and not so much the sorption sequence,

STB40% STB60% – and by the positive control TB100% (P b 0.05) (Fig. 4) with all of them having Kr of 0.97 or higher (Fig. 4); with the exception This increase in the sorption capacity is probably due to the contribution of the Kr of Cu and Zn in STB20%. This exception is possibly due to the of organic matter and increasing pH caused by the amendment (Beesley fact that it contains a low proportion of amendment (Fig. 4), which sug- et al., 2010, Beesley and Marmiroli, 2011; Uchimiya et al., 2011). In the gests that the organic matter content and pH did not increase enough to case of Cu, both the mixtures (STB20%, STB40%, STB60%) and the pure be an effective amendment. amendment TB100% had H-type isotherms indicating that the metal Pb was the metal that was preferably sorbed by the untreated mine has a very high afﬁnity for the metal (Giles et al., 1974)(Fig. 3). This tailing (S), the mixtures (STB20%, STB40%, STB60%) and the positive was demonstrated by the Kr values we obtained, as these were signiﬁ- control TB100% (P b 0.05) (Table 2). In the case of the untreated mine cantly higher than those for S (P b 0.05) (Fig. 4). In the case of Pb, tailing, this behaviour may be due to the fact that it had a very low con-

STB40%, STB60% and TB100% also had H-type isotherms, while the mix- tent of CaCl2-extractable Pb. In the case of the mixtures and the TB100% ture STB20% had an L-type isotherm (Giles et al., 1974)(Fig. 3), with all amendment, the fact that the Pb is preferably sorbed is possibly to the of them having a greater affinity for this metal than mine tailing S. For high affinity of this metal for organic matter. Although CaCl2-extractable Pb, the sorption capacity was estimated using the Kr value for all of Pb content increased with the proportion of amendment, their sorption the mixtures and for the pure amendment it was significantly higher capacity for this metal also increased as their organic matter content than that of S (P b 0.05) (Fig. 4). The Zn behaved differently; as the and pH increased. In S and the STB40% and STB60% mixtures, the mine tailing treated with 20% of the amendment (STB20%) had a sorp- metal that was preferably sorbed after Pb was Cu, and finally Zn tion capacity of practically zero, with nearly all of the Zn remaining in (Table 2). In the case of STB40% and STB60% this was possibly due to the equilibrium solution (Figs. 3 and 4). The mixtures STB40%, STB60% the increase in organic matter and the pH, allowing aggregates to and the pure amendment TB100% had a sorption capacity for Zn form in the mine tailing that retained the Cu. In the case of S, this that was significantly higher than S and STB20% (P b 0.05) (Fig. 4). soil has a high CaCl2-extractable Cu content, and for this reason it will This higher sorption capacity for Zn was reflected in the isotherms, as preferably sorb it before the Zn. The STB20% mixture and positive con- both those of TB100% and STB60% resemble an H-type curve, and for trol TB100% were preferably sorbed after the Pb and Zn, and finally STB40% resemble an L-type curve (Giles et al., 1974) as was the case the Cu (Table 2). with Pb (Fig. 3). This increase in the sorption capacity as a result of adding the amendment could have been favoured by the increased 5. Conclusions pH, caused by adding the amendment comprised of alkaline waste. The Pearson's correlations that were calculated corroborate the sig- The application of an amendment made of a mixture of waste matter nificantly positive correlation amongst soil pH and the Kr for Cu, Pb or and biochar to a mine tailing increased the capacity of the soil to sorb Cu, Zn (r = 0.87, r = 0.77 and r = 0.93 respectively; P b 0.01). Another Pb and Zn. Also, practically all of these metals contained in the

69 R. Forján et al. / Catena 137 (2016) 120–125 125 amendment were retained. It is interesting to note that Zn is the ele- Kabata-Pendias, A., 2001. Trace Elements in Soils and Plants. 3rd ed. CRC Press, (E.E.U.U.). Karami, N., Clemente, R., Moreno-Jiménez, E., Lepp, N.W., Beesley, L., 2011. Efficiency of ment that was sorbed the least from the three elements studied. For green waste compost and biochar soil amendments for reducing lead and copper mo- all of these reasons, the contribution of metals by the amendment bility and uptake to ryegrass. J. Hazard. Mater. 191, 41–48. used is offset by the increase in the sorption capacity of these amend- Kocasoy, G., Güvener, Z., 2009. Efficiency of compost in the removal of heavy metals from the industrial wastewater. Environ. Geol. 57, 291–296. ments for these metals. Therefore, the fact that the amendments contain Natal-da-Luz, T., Ojeda, G., Costa, M., Pratas, J., Lanno, R.P., 2012. Short-term changes of high concentrations of Cu, Pb or Zn is not potentially hazardous to the metal availability in soil. Part I: Comparing sludge-amended with metal-spiked environment. soils. Arch. Environ. Contam. Toxicol. 63, 199–208. Nicholson, F.A., Chambers, B.J., Smith, K.A., 1996. Nutrient composition of poultry manures in England and Wales. Bioresour. Technol. 58, 279– 284. References Paradelo, R., Villada, A., Barral, M.T., 2011. Reduction of the short-term availability of copper, lead and zinc in a contaminated soil amended with municipal solid waste Akala, V.A., Lal, R., 2001. Soil organic carbon pools and sequestration rates in reclaimed compost. J. Hazard. Mater. 188, 98–104. minesoils in Ohio. J. Environ. Qual. 30, 2098–2104. Park, J.H., Choppala, G.K., Bolan, N.S., Chung, J.W., Chuasavathi, T., 2011. Biochar reduces Alberti, G., Cristini, A., Loi, A., Melis, P., Pilo, G., 1997. Copper and lead sorption by different the bioavailability and phytotoxicity of heavy metals. Plant Soil 348, 439–451. fractions of two Sardinian soils. In: Prost, R. (Ed.), Contaminated Soils: Third Int. Conf. Santibañez, C., Ginocchioa, R., Varnero, M.T., 2007. Evaluation of nitrate leaching from on the Biogeochemistry of Trace-elements. INRA Editions, Paris, France ([CD-ROM] mine tailings amended with biosolids under Mediterranean type climate conditions. data/communic/111 PDF). Soil Biol. Biochem. 39, 1333–1340. Asensio, V., Vega, F.A., Sing, B.R., Covelo, F., 2013. Effects of tree vegetation and waste Tejada, M., Parrado, J., Hernández, T., García, C., 2010. The biochemical response to differ- amendments on the fractionation of Cr, Cu, Ni, Pb and Zn in polluted mine soils. ent Cr and Cd concentrations in soils amended with organic wastes. J. Hazard. Mater. Sci. Total Environ. 443, 446–453. 185, 204–211. Beesley, L., Marmiroli, M., 2011. The immobilisation and retention of soluble arsenic, Temminghoff, E.J.M., Van der Zee, S., de Haan, F., 1997. Copper mobility in a copper- cadmium and zinc by biochar. Environ. Pollut. 159, 474–480. contaminated sandy soil as affected by pH and solid and dissolved organic matter. Beesley, L., Moreno-Jiménez, E., Gomez-Eyles, J., 2010. Effects of biochar and greenwaste Environ. Sci. Technol. 31, 1109–1115. compost amendments on mobility, bioavailability and toxicity of inorganic and or- Theodoratos, P., Moirou, A., Xenidi, A., Paspaliaris, I., 2000. The use of municipal sewage ganic contaminants in a multi-element polluted soil. Environ. Pollut. 158, 2282–2287. sludge for the stabilization of soil contaminated by mining activities. J. Hazard. Brown, S., Henry, C., Chaney, R., Compton, H., De Volde, P., 2003. Using municipal biosolids Mater. 77, 177–191. in combination with other residuals to restore metal-contaminated mining areas. Uchimiya, M., Klasson, T., Wartelle, L., Lima, I., 2011. Influence of soil properties on heavy Plant Soil 249, 203–215. metal sequestration by biochar amendment: 1. Copper sorption isotherms and the Canet, R., Pomares, F., Cabot, B., 2007. Composting olive mill pomace and other residues release of cations. Chemosphere 82, 1431–1437. from rural south-eastern Spain. Waste Manag. 28, 2585–2592. Vega, F.A., Covelo, E.F., Andrade, M.L., 2005. Limiting factors for reforestation of mine Covelo, E.F., Vega, F.A., Andrade, M.L., 2007. Heavy metal sorption and desorption capacity spoils from Galicia (Spain). Land Degrad. Dev. 16, 27–36. of soils containing endogenous contaminants. J. Hazard. Mater. 143, 419–430. Vega, F.A., Covelo, E.F., Andrade, M.L., 2008. A versatile parameter for comparing the Giles, C.H., Smith, D., Huitson, A., 1974. A general treatment and classification of the solute capacities of soils for sorption and retention of heavy metals dumped individually adsorption isotherm: I. Theoretical. J. Colloid Interface Sci. 47, 755–765. or together: results for cadmium, copper and lead in twenty soil horizons. J. Colloid Gomes, P.C., Fontes, M.P., da Silva, D.G., Mendonça, S., Netto, A.R., 2001. Selectivity se- Interface Sci. 327, 275–286. quence and competitive adsorption of heavy metals by Brazilian soils. Soil Sci. Soc. Vega, F.A., Covelo, E.F., Andrade, M.L., 2009. Degradation of fuel oil in salt marsh soils Am. J. 65, 1115–1121. affected by the prestige oil spill. J. Hazard. Mater. 169, 36–45. Hackett, A.R., Easton, A., Duff, J.B., 1999. Composting of pulp and paper mill y ash with Weng, L., Temminghoff, E.J.M., Van Riemsdijk, W.H., 2001. Contribution of individual sor- wastewater treatment sludge. Bioresour. Technol. 70, 217–224. bents to the control of heavy metal activity in sandy soil. Environ. Sci. Technol. 35, Harter, R.D., Naidu, R., 2001. An assessment of environmental and solution parameter 4436–4443. impact on trace-metal sorption by soils. Soil Sci. Soc. Am. J. 65, 597–612. Zanuzzi, A., Arocena, J.M., van Mourik, J.M., Faz, A., 2009. Amendments with organic and Houba, V.J.G., Temminghoff, E.J.M., Gaikhorst, G.A., Van Vark, W., 2000. Soil analysis industrial wastes stimulate soil formation in mine tailings as revealed by micromor- procedures using 0,01 M calcium chloride as extraction reagent. Commun. Soil Sci. phology. Geoderma 154, 69–75. Plant Anal. 3, 1299–1396. Illera, V., Walter, I., Souza, P., Cala, V., 2000. Short-term effects of biosolid and municipal solid waste applications on heavy metals distribution in a degraded soil under a semi-arid environment. Sci. Total Environ. 255, 29–44.

2.3 Contributions of a compost-biochar mixture to the metal sorption capacity of a mine tailing

Environ Sci Pollut Res (2016) 23:2595–2602 DOI 10.1007/s11356-015-5489-0

RESEARCH ARTICLE

Contributions of a compost-biochar mixture to the metal sorption capacity of a mine tailing

R. Forján1 & V. Asensio2 & A. Rodríguez- Vila1 & E. F. Covelo1

Received: 21 May 2015 /Accepted: 21 September 2015 /Published online: 3 October 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract One technique applied to restore degraded or con- Keywords Compost . Mine tailing . Mine . Metal . Soil taminated soils is to use amendments made of different types remediation . Biochar . Sorption of waste materials, which in turn may contain metals such as Cu, Pb and Zn. For this reason, it is important to determine the capacity of the soil to retain these materials, and to compare Introduction the sorption capacity between an amended soil and another unamended soil. The aim of this study was to determine the Mine soils are highly degraded at both the physical and chem- mobility and availability of these metals in the soil after ap- ical level, which inhibits normal soil functions, such as plying the amendment, and how it affected the soil’ssorption supporting plant growth (Xia and Cai 2002). Mine tailing capacity. Sorption isotherms were compared with the empiri- are a source of pollution that affects the immediate and sur- cal models of Langmuir and Freundlich to estimate the sorp- rounding areas. This is due to the contamination of under- tion capacity. The overall capacity of the soils to sorb Cu, Pb ground waters as a result of acid runoff from the mine, or or Zn was evaluated as the slope Kr. The amendments used in atmospheric pollution caused by fine particles that are carried this study were a mixture made of compost and biochar in off into the air. One of the main impacts that mining has on different proportions (20, 40, 60, 100 %), which were applied soils is their high concentration of metals. Also, some soil to the mine tailing from a settling pond from a copper mine. properties are affected, such as acidity and organic matter The mine tailing that were amended with the mixture of com- content (Akala and Lal 2001;Vegaetal.2005; Santibañez post and biochar had a higher sorption capacity than the mine et al. 2007; Zanuzzi et al. 2009; Asensio et al. 2013) which tailing from the unamended pond, and their sorption isotherms can affect carbon sequestration in the soil. It is important to had a greater affinity towards Cu, Pb and Zn than the mine understand these metallic elements and the ease with which tailing that was studied. Therefore, the results obtained show they migrate through the mine tailing. that adding a mixture of compost and biochar favours the Current approaches used to remedy soil contamination retention of Cu, Pb and Zn in mine tailing. caused by metals are often based on the immobilisation of the metals in situ, leaving them in less bioavailable forms (Kiikkilä et al. 2002). Using amendments produced by Responsible editor: Philippe Garrigues composting to increase the organic content of degraded soils is a classic technique which is currently being perfected by * R. Forján using higher-quality compost. Contributing organic matter to [email protected] these types of degraded soils is important, as this leads to a decrease in the available metal content due to the organic 1 Department of Plant Biology and Soil Science, Faculty of Biology, matter being capable of forming strong bonds with the metals University of Vigo, As Lagoas-Marcosende, as thereby retain them strongly in the soil (Kabata-Pendias 36310 Vigo, Pontevedra, Spain 2001). However, on occasions, the waste matter used to make 2 Department of Plant Nutrition, CENA, University of São Paulo compost intended to be used as a soil amendment contains (CENA-USP), Av. Centenário 303, 13400-970 Piracicaba, SP, Brazil contaminating materials, which means that many of them

75 2596 Environ Sci Pollut Res (2016) 23:2595–2602 cannot be used on any kind of soil. It is also necessary to check Understanding metal sorption processes can provide very the soil/amendment proportions used in order to balance out valuable information in order to know how amendments really their intended benefits with any potential contamination prob- work once they have been applied to the soil. Therefore, the lems (Paradelo et al. 2011). Also, using these organic amend- aim of this study was to compare the sorption of Cu, Pb and ments can re-distribute metals from the exchangeable and Zn in mine tailing from a copper mine, after applying an water-soluble phase to less available organic or residual frac- amendment made of compost and biochar. tions. This reduction in the bioavailability of metals is both the result of increasing the sorption of these chemical elements on surfaces with colloidal particles, and of the formation of stable Material and methods complexes with the organic matter (Pérez-Esteban et al. 2012). Adding organic matter to soil also improves the physical and Study area and experiment design chemical properties of soil (Gadepalle et al. 2007;Illeraetal. 2000), contributes C and essential nutrients (e.g. N, P), which The sample zone is in the mine in Touro (Northwest Spain; Lat/ then become part of the biochemical cycle of the resulting Lon (Datum ETRS89): 8° 20′ 12.06″ W 42° 52′ 46.18″ N). The ecosystem. However, the C in compost is quickly lost due to climate in this zone is Atlantic (oceanic) with precipitation its relatively low recalcitrance. These composts are made using reaching 1886 mm per year (with an average of 157 mm per materials which in theory do not have any metal added to them, month) and a mean daily temperature of 12.6 °C. The average of or which have a minimum metal content, as their production relative humidity is 77 % (AEMET by Spanish Meteorological process must comply with a series of legal requirements, and Agency). For this study, mine tailing was chosen from the the waste materials that can be used for this purpose are also mine’s settling pond (S). The mine tailing (S) was comprised subject to regulations (Decision 573-2001 EC, Spanish Law of waste material resulting from the flotation of sulphides during 22/2001). Different studies have shown that the application of copper processing. The compost used consisted of horse and amendments made from waste matter reduces the available rabbit manure mixed with grass cuttings, fruit and seaweed metal content in the soil (Brown et al. 2003; Natal-da-Luz (C). The biochar (B) was made of biomass from areas invaded et al. 2012). Amongst other reasons, this is because the organic by A. dealbata in the north of Portugal (temperature 450 °C, matter in the amendments interacts with the metal ions to form time 8 h). The mine tailing was treated with the amendment insoluble complexes (Martínez et al. 2007). These studies are CB, made with 97 % compost (C) combined with 3 % biochar based on an understanding of how the available metal content (B). The amendment (CB) and the mine tailing (S) were mixed in the soil varies, but not on how these metals behave in the soil (SCB) at 20, 40 and 60 % of CB. All mixtures were made w/w. once the amendments have been applied. The amendment CB100 % and the mine tailing S100 % were One way of reinforcing the effect of compost as an amend- the control samples. All samples were incubated in glass vials, in ment is by mixing it with biochar, although this is currently a dark to field capacity for 1 month, adding distilled water period- costly process (Beesley et al. 2010a). Biochar is obtained by ically to maintain constant weight. After that, samples were dried burning biomass (both plant waste and animal remains) in a and sieved with a sieve with a pore diameter of 2 mm. substoichiometric ratio or without oxygen in pyrolytic ovens. Like other amendments made of waste, biochar contributes Chemical analyses organic matter, especially carbon and nitrogen. But unlike these, the waste material used to make biochar usually has a pH was determined with a pH electrode in 1:2.5 soil:water low or negligible content of trace elements (Beesley et al. extracts (Porta 1986). The phytoavailable content of Cu, Pb

2010a, b, Fellet et al. 2011,Parketal.2011). However, as and Zn was extracted with 0.01 M CaCl2 in soil solution an added benefit, the C and N they contribute is much more (Houba et al. 2000). Quasitotal metal contents were extracted recalcitrant. Different studies have shown how biochar can with aqua regia by acid digestion in a microwave oven reduce the available contents in metals such as Cu, Pb and (Milestone ETHOS 1, Italy) and analyzed in an ICP-OES Zn and increase the pH of the soil (Beesley et al. 2010b; (PerkinElmer Optima 4300 DV). Soil total carbon (TC) was Beesley and Marmiroli 2011;Parketal.2011). Biochar can determined in a solid module (Shimadzu SSM-5000, Japan) reduce the availability of metals due to its complexing metal coupled with a TOC analyser (Shimadzu TNM-1, Japan). ions on its surface (Beesley et al. 2010a). The biochar selected for this study was made from Acacia Sorption experiment and construction of isotherms dealbata Link. This is a shrub that belongs to the legume family (Fabaceae) and is considered to be an invasive species The sorption capacity was evaluated following the batch in the Iberian Peninsula. The use of biochar made of method of Alberti et al. (1997) and Gomes et al. (2001)mod- A. dealbata as an amendment therefore has two functions: ified by Harter and Naidu (2001). They used single-metal recovering the soil and using waste from an invasive species. solutions of Cu2+,Pb2+ and Zn2+ nitrate in 0.03, 0.05, 0.08,

76 Environ Sci Pollut Res (2016) 23:2595–2602 2597

−1 0.1 and 0.5 mmol L in 0.01 M NaNO3 as background elec- a It is the Kr parameter. It is the slope of the line. It is a trolyte (Vega et al. 2009). Tri-metal equimolar solutions of dimensionless parameter. If a≈1 high affinity for the Cu2+,Pb2+ and Zn2+ as nitrates were also prepared with the metal. same background electrolyte (0.01 M NaNO3). Then, 25 mL of the mono-metallic metal solutions were added to 1.5 g of soil samples in polyethylene tubes and shaken for 24 h at 25 °C. After centrifugation during 10 min at 3000 rpm, the supernatant was filtered through Whatman 42 paper (pore size Statistical analyses 0.45 μm). Filtered supernatants were analyzed for Cu, Pb and Zn concentrations by ICP-AES (PerkinElmer Optima 4300 All of the analytical determinations and sorption isotherms DV). The amount of metal sorbed is defined as the difference were performed in triplicate. The data obtained were statisti- between the concentration added (from sorption solution) and cally treated with the program SPSS version 19.0 for the concentration in the solution after equilibration (24 h shak- Windows. Analysis of variance (ANOVA) and test of homo- ing) with the soil. To minimize the effect of the background geneity of variance were carried out. In case of homogeneity, a electrolyte, the sorbed concentrations were corrected by sub- post hoc least significant difference (LSD) test was carried traction of the values obtained in additional experiments with out. If there was no homogeneity, Dunnett’sT3testwasper- sorption solution composed only by 0.01 M NaNO3. formed. An independent t test was carried out in order to For each metal and treatment sorption, isotherms were con- compare all of the soils when the data were not parametric. structed by plotting the sorbed metal concentration (μmol per g A correlated bivariate analysis was also carried out with data of dry soil sample) against the concentration of the metal in from all of the soil samples. solution at equilibrium (μmol L−1). The obtained isotherms were compared to the models of Langmuir (1) and Freundlich (2) and with the types of curve described by Giles et al. (1974). The overall capacity of the soils to sorb Cu, Pb or Zn was Results evaluated as the slope Kr (3) according to Vega et al. (2008). Characteristics of the mine tailing (S), compost (C) 1. Langmuir model: and biochar (B) = ¼ β = þ x m CKL L 1 KLC The mine tailing (S) had an acidic pH, while the compost and the biochar used (C and B, respectively) had significantly x Quantity of material sorbed (μmol) higher pH than S (Table 1). The biochar and the compost also m Quantity of sorben (g) had significantly higher concentration of total carbon (TC) C Solution concentration equilibrium (μmol L−1) than the mine tailing. The highest concentration of both K Langmuir constant (L μmol L−1) L quasitotal and CaCl -extractable Cu was observed in the mine β High sorption capacity (μmol g−1) 2 L tailing, and the compost and biochar had significantly lower concentrations. The highest concentrations of quasitotal Pb 2. Freundlich model: was observed in the compost (Table 1). The highest Log x=m ¼ 1=nlogCþ log K F Table 1 Characteristics of mine tailing (S), compost (C) and biochar x Quantity of material sorbed (μmol) (B) m Quantity of sorben (g) Mine tailing (S) Compost (C) Biochar (B) C Solution concentration equilibrium (μmol L−1) −1 KF Freundlich constant (L g ) pH 2.62±0.01c 6.25±0.04b 9.44±001a n Dimensionless. n› 1, sorption is favourable C(mgkg−1) u.d 274±2.6b 672±5.5a Cu Quasitotal 636±50a 162±4.1b 27.7±0.9c 3. −1 Kr Pb(mg kg ) 8.21±0.8b 39.5±0.7a u.d Zn 63.7±4.1b 309±4.3a 64.5±0.7b

It is a line of type y=ax Cu CaCl2-extractable 138±13a 0.98±0.1b u.d −1 − (mg kg ) y Quantity of sorbent (μmol g 1) Pb 0.28±0.1a 0.17±0.1a u.d x Amount of added metal or metals per gram of soil Zn 2.41±0.01b 7.25±0.1a 1.31±0.1c (deduced from the concentration of the solution with For each row, different letters in different samples means significant dif- which were treated) versus sorbed amount of each of the ferences (n=3, ANOVA; P<0.05). u.d. undetectable level. Typical devi- − metals (μmol g 1) ation is represented by ±

77 2598 Environ Sci Pollut Res (2016) 23:2595–2602

concentration of quasitotal and CaCl2-extractable Zn was observed in the compost (Table 1).

Characteristics of the mine tailing (S), amended mine tailing (SCB20 %, SCB40 %, SCB60 %) and the compost + biochar positive control (CB100 %)

All of the samples containing compost + biochar (with a compost + biochar:soil proportion of 20, 40 and 60 %) had a significantly higher pH than the S. The sample with the highest pH was the CB100 % (Fig. 1). Here, we see that pH increased as the proportion of added amendment increased the quasitotal concentration of Cu was significantly higher in the mine tailing (S) and in the 20 % amended mine tailing (SCB20 %) in comparison with the other samples (Fig. 2a). In the case of Pb, the positive control (CB100 %) and the SCB60 % mixture had a higher quasitotal content than the rest (Fig. 2a). The quasitotal content of Zn showed an upward trend as the percentage of amendment applied to the mine tailing was increased, with the lowest content in S and the highest value in the positive control CB100 % (Fig. 2a). The

CaCl2-extractable Cu content revealed significant differences, with the highest content in S (Fig. 1b). In the mixtures SCB20, SCB40, SCB60 and CB100 %, there were no significant differences between them in terms of the CaCl2-extractable Cu Fig. 2 a Quasitotal and b CaCl2-extractable Cu, Pb and Zn content. The CaCl2-extractable Pb content in S was signifi- concentrations. For each row, different letters in different samples cantly higher than that of the mine tailing amended with the means significant differences (n =3, ANOVA; P <0.05). u.d. different proportions (SCB20, 40 and 60 %) and CB100 %. undetectable level. Error bars represent typical deviation

The CaCl2-extractable Pb content in the SCB20, SCB40 and SCB60 % and in the positive control CB100 % was undetect- Sorption capacity of Cu, Pb and Zn by the amended able. Also, the mixtures SCB20, 40 and 60 % had a signifi- and unamended mine tailing cantly higher CaCl2-extractable Zn in comparison to S and CB100 %. Figure 3a–c show the individual sorption isotherms of Cu, Pb and Zn, except for those of the unamended mine tailing (S), whose sorption capacity was zero for all three elements. As may be seen in the case of Cu, the positive control CB100 % (100 % compost and biochar) and the mixtures SCB60 % and SCB40 % have a high affinity for this metal. The sorption isotherms of Cu for the different proportions and the positive control do not fit any of the curves proposed by Giles et al. (1974)(Fig.3a). The sorption isotherms for Pb have an initially steep slope for CB100, SCB60 and SCB40 %, which have the greatest affinity for this metal (Fig. 3b). The slope of the sorption isotherm for SCB20 % was less steep, although it did have a higher affinity for Pb than soil S. The Pb sorption isotherms for SCB40, SCB60 and CB100 % fit the H-type curves proposed by Giles et al. (1974). The samples CB100, SCB60 and SCB40 % have the greatest affinity for Zn of all of the samples based on the Fig. 1 pH values of the different treatments of the mine tailing. For each row, different letters in different samples means significant differences isotherms that were created. The isotherm for Zn for the (n=3, ANOVA; P<0.05). Error bars represent typical deviation SCB20 % mixture shows that practically all of the metal

78 Environ Sci Pollut Res (2016) 23:2595–2602 2599

SCB60 % and the positive control CB100 % fit the Langmuir model (Table 2). TSoil S, the mixture SCB20 and CB100 %, fit the Freundlich model (Table 2). The Zn isotherms for S and CB100 % fit the Freundlich model (Table 2). In the case of Zn, both the soil, the mixtures and the positive control fit the Langmuir model (Table 2). Having observed that not all of the isotherms were a close fit to the Langmuir and Freundlich models, the distribution

coefficient Kr was calculated in order to estimate the sorption capacity (Vega et al. 2008). As shown in Table 2,theKr obtained for Cu showed that S has a zero sorption capacity for this metal. In contrast, the positive control (CB100 %) and amended soil SCB60 % had a significantly higher sorption capacity for Cu than SCB20 and SCB40 % (Table 2). The

Kr obtained for Pb indicates that there are no significant differences between the sorption capacity for this metal amongst all of the samples that contain amendments, including the pure amendment (CB100 %). The unamended control sample (S) did have a sorption capacity for Pb, although significantly

lower than the Kr for the amended soil and the positive control (Table 2). The Kr obtained for Zn indicated that soil S has a zero sorption capacity for Zn. All of the mixtures SCB20, SCB40 and SCB60 % and the positive control CB100 % had a significantly higher sorption capacity for Zn than S (Table 2). Table 2 shows a comparison of the sorption preferences of mine tailing (S), the mixtures (SCB20 %, SCB40 %, SCB60 %) and the positive control (CB100 %) for Cu, Pb

and Zn based on the Kr calculated. S and SCB40 % have a similar pattern, where it can be seen that they preferably sorb Pb. Out of the samples, SCB20 and SCB60 % have the same sorption preference for the three metals studied. The positive control preferably sorbs Pb and Cu. In general terms, the metal with the lowest sorption preference is Zn.

Fig. 3 Sorption isotherms of a Cu, b Pb and c Zn Discussion

General characteristics of the studied samples remains in the equilibrium solution, on the contrary to the situation with this same mixture for Cu and Pb (Fig. 3). The pH value of the mine tailing from the copper settling pond None of the isotherms for the different proportions or the (S) amended with different mixtures of compost and biochar positive control fit any of the curves proposed by Giles et al. (SCB20 %, SCB40 %, SCB60 %) constantly increased as the (1974). proportion of amendment applied increased. This increase in On attempting to estimate the sorption capacity for Cu, Pb the pH value is due to the alkaline pH of the waster matter and Zn of the samples that were studied according to the used to make the compost (horse and rabbit manure, plant Langmuir and Freundlich adjustments (Freundlich 1926; trimmings and seaweed), and of the biochar used. This in- Langmuir 1918), it was found that not all of the isotherms crease in the pH is important because it reduces the mobility fitted them (Table 2). In the case of the sorption isotherms of of the metals in the soil (Park et al. 2011;Wengetal.2001), Cu, only those of soil S and the mixture SCB40 % fitted the and also means that there are more nutrients in an available Freundlich model (Table 2). The Cu isotherms of the other form. mixtures (SCB20, SCB40, SCB60 and CB100 %) fit the The CaCl2-extractable concentrations of Cu, Pb and Zn Freundlich model (Table 2). In the case of Pb, only the mixture varied greatly depending on the metal and the percentage of

79 2600 Environ Sci Pollut Res (2016) 23:2595–2602

Table 2 Fitting sorption S SCB20 % SCB40 % SCB60 % CB100 % isotherms and Kr

Langmuir Cu R2 0 0.111±0.001 0.173±0.001 0.191±0.001 0.248±0.002 β –– – – – Pb R2 0 0.066±0.001 0.034±0.002 1 1 β –– – –– Zn R2 0 0.207±0.002 0.392±0.003 0 0.973±0.001 β –– – – 9.633±0.001 Freundlich Cu R2 0 0.985±0.002 0.973±0.001 0.981±0.001 0.974±0.003

KF – 0.541±0.001d 0.654±0.01c 0.990±0.020b 1.282±0.011a Pb R2 0 0.913±0.021 0.512±0.011 0.417±0.031 0.941±0.001

KF – 3.332±0.042a 2.481±0.031b 1.637±0.001d 1.859±0.015c Zn R2 0 0.972±0.001 0.996±0.001 0.949±0.003 0.959±0.001

KF – 0.901±0.001d 3.224±0.041c 4.319±0.032b 8.014±0.075a 2 Kr Cu R 0 1 1 0.999±0.001 1

Kr 0c 0.981±0.004b 0.986±0.001ab 0.991±0.001a 0.994±0.001a Pb R2 01 1 1 1

Kr 0c 0.996±0.001a 0.999±0.001a 0.996±0.001a 0.951±0.005a Zn R2 0 1 1 1 0.999±

Kr 0b 0.991±0.001a 0.995±0.001a 0.994±0.001a 0.971±0.021a

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). Typical deviation is represented by ± the amendment applied to the soil (Fig. 2b). Soil S had a amounts of available Zn in soil become toxic to plants; in leaf, significantly higher CaCl2-extractable Cu content due to the it induces visible toxicity symptoms such as necrosis and chlo- degradation of the starting material in the area (almost exclu- rosis. In the roots, it decreases the elongation of main root and sively consisting of amphibolites) and its acidic pH. As a increasing formation of lateral roots. Other effect includes result of being amended with the mixture of compost and decrease in tissue water content and changes in tissue biochar, the CaCl2-extractable Cu content decreased due to (Romeo et al. 2014). the increased pH and concentration of organic matter (Park et al. 2011; Pérez-Esteban et al. 2012). The CaCl2-extractable Sorption capacity of the studied samples for Cu, Pb Pb content was also higher in the unamended soil, and was and Zn practically reduced to undetectable levels in all of the amended samples. This is also due to the increased pH and The mine tailing without any type of treatment (S) has a zero organic matter in the amended soils. Zn was the metal with the sorption capacity for Cu and Zn, and a very low sorption highest concentration in CaCl2-extractable form. The mixtures capacity for Pb (data not shown). This may be due to the SCB20, 40 and 60 % had the highest concentration, in all low organic matter content and highly acidic pH of this soil. likelihood due to the high initial concentration of quasitotal This very low pH is a limiting factor for plant growth (Buol Zn in the compost, and the fact that the decrease in the pH after et al. 1975), and by being so acidic, means that in this type of mixing it with the mine soil caused the Zn to pass to the soil, the metals are in an available form (Table 1). (Park et al. mobile phase. This is also due to the fact that Zn tends to form 2011;Wengetal.2001). In fact, the CaCl2-extractable content external sphere complexes with the organic matter, which are of Cu and Pb is significantly higher in the mine tailing than in very weak and which as a result of variations in the pH may the amended mine tailing (Fig. 2b). In the case of Cu, the cause the Zn to be released (McBride et al. 1997). Also, those percentage of CaCl2-extractable content in comparison to the with the lowest CaCl2-extractable Zn were S and the quasitotal content goes from 21 % in S to less than 1 % in the CB100 %. In the case of CB100 %, the content was signifi- mixtures applied. In the case of Pb, the percentage of CaCl2- cantly lower than the rest of the amendments, as although the extractable content compared to the quasitotal content is 0 % compost contains Zn, the increased pH of CB100 % helps to in the case of the mine tailing amended with the different fix this metal. Previous studies have shown that soil pH plays percentages of amendment. This indicates how effective this a crucial role in metal solubility (Park et al. 2011;Wengetal. amendment is at retaining these metals. In the case of Zn, the

2001). It is important to take into account that Zn is the metal percentage of CaCl2-extractable content in comparison to the that has the least affinity in the sorption sites in the soil. High quasitotal content in the mine soil amended in different

80 Environ Sci Pollut Res (2016) 23:2595–2602 2601 proportions is 10, and 4 % in S. This is due to the high Zn extractable contents compared to the total content of Cu, Pb content of the compost, and the fact that this metal forms weak and Zn is 21, 0.04 and 3.70 % respectively, while in the case of or unstable complexes with the organic matter (McBride et al. the SCB60 %, for example, the content for the same metals 1997). Even so, the concentrations of Zn in phytoavailable (Cu, Pb and Zn) is 0.05, 0 and 8.36 % respectively. This shows form are low in all of the cases comparing with the Kelly that despite contributing metallic elements, the amendment Indices (<250 mg kg−1). would be beneficial, as it has a high sorption capacity for Sorption isotherms had a better adjustment to the metals, contributes organic matter and increases the pH of Freundlich model than to Langmuir one. It can be explained mine tailing. because some of the assumptions of the Langmuir model are Recovering mine tailing by applying this organic amend- that there is no interaction amongst sorbate molecules, that ment, consisting of a combination of an amendment made of binding at one site does not affect binding at another and that compost and with biochar, significantly increases the sorption each binding site can bind just one sorbate molecule capacity of Cu, Pb and Zn in all of the proportions used (Bmonolayer^ sorption), the parameter β of the model (SCB20, SCB40 and SCB60 %), being most effective in those reflecting the number of binding sites and hence the maximum in which SCB40 % and SCB60 % were applied (Table 2). amount of sorbate that can be sorbed (Covelo et al. 2008). This is because the higher the proportion of amendment in Despite of the not adjustment of the obtained sorption iso- the soil, the higher the contribution of carbon and increase in therms to Langmuir and Freundlich models, we can highlight the pH. that the KF value is higher in the treated samples than in the original mine tailing (Table 2). According to the Kr value, sorption capacity of S increased Conclusions significantly once it had been amended with the different mixtures used in this study (SCB20 %, SCB40 % SCB60 %) and The application of an amendment made of a mixture of com- with the positive control CB100 % (P<0.05) (Table 2). This post and biochar to a mine soil increased the capacity of the amendment increases the affinity of the mine tailing for the soil to sorb Cu and Pb. Also, practically all of these metals metals, as may be seen by the type of curve and the sorption contained in the amendment were retained. It is interesting to capacity according to the values of Kr.Thisincreaseinthe note that Zn is the element that was sorbed the least from the sorption capacity of the mine tailing for Cu, Pb and Zn due three elements studied. For all of these reasons, the contribu- to the contribution of amendment may have been favoured by tion of metals by the amendment used is offset by the increase the increased pH caused by adding the waste materials used, in the sorption capacity of these amendments for these metals. with an alkaline pH, and by the increased concentration of Therefore, the fact that the amendments used contain high organic matter, which interacts with metal ions to form insol- concentrations of Cu, Pb or Zn is not potentially hazardous uble complexes (Martínez et al. 2007). It is well known that to the environment. high pH favours metal sorption in soils. The calculated Pearson’s correlations corroborate this by the significantly positive correlation amongst soil pH and the Kr for Cu, Pb or Zn (r=0.73, P<0.01; r=0.63, P<0.05 and r=0.71, References P<0.01). Another reason why the sorption capacity increased in S is possibly due to the increased amount of organic matter, Akala VA,Lal R (2001) Soil organic carbon pools and sequestration rates after amending the soil in different proportions (SCB20 %, in reclaimed minesoils in Ohio. J Environ Qual 30:2098–2104 SCB40 %, SCB60 %); as it has been shown that soils with a Alberti G, Cristini A, Loi A, Melis P, Pilo G (1997) Copper and lead higher organic matter content have a greater capacity to retain sorption by different fractions of two Sardinian soils. In: Prost R (Ed.) Contaminated soils: third int. conf. on the biogeochemistry metals (Covelo et al. 2007;Parketal.2011; Pérez-Esteban of trace-elements. [CD-ROM] data/ communic/111 PDF. INRA et al. 2012). Editions,Paris,France The contribution of metallic elements from the amendment Asensio V, Vega FA, Sing BR, Covelo F (2013) Effects of tree vegetation applied, especially the Zn provided by the compost, would be and waste amendments on the fractionation of Cr, Cu, Ni, Pb and Zn in polluted mine soils. Sci Total Environ 443:446–453 mitigated by the high sorption capacity of the amendment. Beesley L, Marmiroli M (2011) The immobilisation and retention of The percentages of CaCl2-extractable contents in comparison soluble arsenic, cadmium and zinc by biochar. Environ Pollut 159: to the total Cu, Pb and Zn contents is lower in the soil 474–480 amended with the different amendments (SCB20 %, Beesley L, Moreno-Jiménez E, Gomez-Eyles JC, Harris E, Robinson B, ’ SCB40 %, SCB60 %) and in the positive control Sizmur T (2010a) A review of biochars potential role in the remediation, revegetation and restoration of contaminated soils. Environ (CB100 %) in comparison to the untreated soil (S), except Pollut 159:3269–3282 for Zn, whose percentages are low, but still higher than those Beesley L, Moreno-Jiménez E, Gomez-Eyles J (2010b) Effects of biochar of soil S. For example, in S, the percentages of CaCl2- and greenwaste compost amendments on mobility, bioavailability

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and toxicity of inorganic and organic contaminants in a multi- Martínez CE, Bazilevskaya KA, Lanzirotti A (2007) Zinc coordination to element polluted soil. Environ Pollut 6:2282–2287 multiple ligand atoms in organic-rich surface soils. Environ Sci Brown S, Henry C, Chaney R, Compton H, De Volde P (2003) Using Technol 40:5688–5695 municipal biosolids in combination with other residuals to restore McBride MB, Suavé S, Hendershot W (1997) Solubility control of Cu, metal-contaminated mining areas. Plant Soil 249:203–215 Zn, Cd and Pb in contaminated soils. Eur J Soil Sci 48:337–346 Buol SW, Sanchez PA, Cate RB, Granger MA (1975) Soil fertility capa- Natal-da-Luz T, Ojeda G, Costa M, Pratas J, Lanno RP, Van Gestel CAM, bility classification. In: Bornemizza E, Alvarado A (eds) Soil Sousa JS (2012) Short-term changes of metal availability in soil. Part Manag. in Trop. Amer. NCS University, Raleigh, pp 126–146 I: comparing sludge-amended with metal-spiked soils. Arch Environ Covelo EF, Vega FA, Andrade ML (2007) Heavy metal sorption and Contam Toxicol 63:199–208 desorption capacity of soils containing endogenous contaminants. Paradelo R, Villada A, Barral MT (2011) Reduction of the short-term J Hazard Mater 143:419–430 availability of copper, lead and zinc in a contaminated soil amended – Covelo EF, Vega FA, Andrade ML (2008) Sorption and desorption of Cd, with municipal solid waste compost. J Hazard Mater 188:98 104 Cr, Cu, Ni, Pb and Zn by a Fibric Histosol and its organo-mineral Park JH, Choppala GK, Bolan NS, Chung JW, Chuasavathi T (2011) fraction. J Hazard Mater 159:342–347 Biochar reduces the bioavailability and phytotoxicity of heavy – Decisión del Consejo de la Comunidad Europea (2001) Decisión del metals. Plant Soil 348:439 451 Consejo 573/2001 Pérez-Esteban J, Escolástico C, Masaguer A, Moliner A (2012) Effects of Fellet G, Marchiol L, Delle Vedove G, Peressotti A (2011) Application of sheep and horse manure and pine bark amendments on metal distri- biochar on mine tailings: effects and perspectives for land reclama- bution and chemical properties of contaminated mine soils. Eur J – tion. Chemosphere 83:1262–1267 Soil Sci 63:733 742 Porta J (1986) Técnicas y experimentos en Edafología. Collegi Oficial Freundlich H (1926) Colloid and capillary chemistry. Methuen, London D’Enginyers Agronoms de Catalunya, Barcelona Gadepalle V, Ouki SK, Van Herwijnen R, Hutchings T (2007) Romeo S, Francini A, Ariani A, SebastianI L (2014) Phytoremediation of Immobilization of heavy metals in soil using natural and waste ma- Zn: identify the diverging resistance, uptake and biomass production terials for vegetation establishment on contaminated sites. J Soil behaviours of poplar clones under high zinc stress. Water Air Soil Contam 16:233–251 Pollut 225:1813. doi:10.1007/s11270-013-1813-9 Giles CH, Smith D, Huitson A (1974) A general treatment and classifi- Santibañez C, Ginocchioa R, Varnero MT (2007) Evaluation of nitrate cation of the solute adsorption isotherm: I. Theoretical. J Colloid leaching from mine tailings amended with biosolids under Interface Sci 47:755–765 Mediterranean type climate conditions. Soil Biol Biochem 39: Gomes PC, Fontes MPF, da Silva DG, Mendonça ES, Netto AR (2001) 1333–1340 Selectivity sequence and competitive adsorption of heavy metals by Vega FA, Covelo EF, Andrade ML (2005) Limiting factors for reforesta- – Brazilian soils. Soil Sci Soc Am J 65:1115 1121 tion of mine spoils from Galicia (Spain). Land Degrad Dev 16:27– Harter RD, Naidu R (2001) An assessment of environmental and solution 36 parameter impact on trace-metal sorption by soils. Soil Sci Soc 65: Vega FA, Covelo EF, Andrade ML (2008) A versatile parameter for com- – 597 612 paring the capacities of soils for sorption and retention of heavy Houba VJG, Temminghoff EJM, Gaikhorst GA, Van Vark W (2000) Soil metals dumped individually or together: results for cadmium, copper analysis procedures using 0,01M calcium chloride as extractation and lead in twenty soil horizons. J Colloid Interface Sci 327:275– – reagent. Soil Sci Plant Anal 31:1299 1396 286 Illera V, Walter I, Souza P, Cala V (2000) Short-term effects of biosolid Vega FA, Covelo EF, Andrade ML (2009) Degradation of fuel oil in salt and municipal solid waste applications on heavy metals distribution marsh soils affected by the Prestige oil spill. J Hazard Mater 169:36– in a degraded soil under a semi-arid environment. Sci Total Environ 45 255:29–44 Weng L, Temminghoff EJM, Van Riemsdijk WH (2001) Contribution of Kabata-Pendias A (2001) Trace elements in soils and plants, 3rd edn. individual sorbents to the control of heavy metal activity in sandy. CRC Press, Boca Raton, EEUU Soil Environ Sci Technol 35:4436–4443 Kiikkilä O, Pennanen T, Perkiömäki J, Derome J, Fritze H (2002) Organic Xia H, Cai X (2002) Ecological restoration technologies for mined lands. material as a copper immobilising agent: a microcosm study on Chin J Appl Ecol 13:1471–1477 remediation. Basic Appl Ecol 3:245–253 Zanuzzi A, Arocena JM, van Mourik JM, Faz A (2009) Amendments Langmuir I (1918) The adsorption of gases on plane surfaces of glass, with organic and industrial wastes stimulate soil formation in mine mica and platinum. J Am Chem Soc 40:1361–1403 tailings as revealed by micromorphology. Geoderma 154:69–75

Justificación y objetivos

88 Justificación y objetivos

3. Justificación y objetivos

Justificación y objetivos

88 Justificación y objetivos

3. Justificación y objetivos

En los trabajos previos presentados al comienzo de esta memoria, hemos observado que los tecnosoles aplicados como enmienda única sobre el suelo de la balsa de decantación de la mina de Touro (A Coruña) mejoran características de los mismos como pH, capacidad de intercambio catiónico, carbono o nitrógeno; pero pueden aumentar el contenido de algunos elementos potencialmente tóxicos (Forján et al., 2014). Sin embargo, si combinamos el tecnosol con biochar, su capacidad para fijar estos metales aumenta (Forján et al., 2016a). Por otro lado, observamos que el aporte de metales por parte compost también disminuye al aplicarlo con biochar y aumenta su capacidad de inmovilización de elementos potencialmente tóxicos (Forjan et al., 2016b).

La mayoría de los experimentos desarrollados por otros autores sobre este tema se han llevado a cabo solamente en macetas o pequeñas cantidades en el laboratorio, estudiando solo el horizonte superficial del suelo y solo con una enmienda (Hattab et al., 2014, Park et al., 2016, Walker et al., 2003, Younis et al., 2016). Actualmente las diferentes enmiendas que se aplican al suelo se empiezan a combinar con el cultivo de plantas fitorremediaroras (Brassica Juncea L.). Brassica juncea L. ha sido utilizada con éxito en la fitorremediación de suelos contaminados por EPTs (Lombi et al., 1999; Do Nascimento et al., 2006; Rodríguez-Vila et al., 2014).

Los metales objeto de estudio en esta tesis fueron el Cu, Pb, Ni y Zn. El cobre fue escogido debido a que es el metal predominante en el suelo objeto de estudio en esta tesis y que, debido a la alteración de este suelo, se encuentra de forma fitodisponible en altas concentraciones (Rodríguez-Vila et al., 2014). Los otros metales estudiados Pb, Ni y Zn predominan en las enmiendas elaboradas con residuos, algunos de los cuales son restos de industrias agroalimentaria, lodos de depuradora, arenas de plantas depuradoras (Amir et al. 2005; Canet et al., 2007; Pérez-Esteban et al., 2014, Smith 2009; Travar et al. 2015; Weber et al. 2007). Además, altas cantidades de estos EPTs pueden reducir la producción vegetal debido al riesgo de biomagnificación y bioacumulación en la cadena alimentaria (Rahman et al., 2013).

Por todo esto, los objetivos principales de esta tesis son:

1. Evaluar el efecto de diferentes enmiendas tanto por separado como combinadas tanto entre sí como con Brassica juncea L.

2. Evaluar cuál es el tratamiento que mejor actúa en la recuperación de un suelo de mina de las características de la mina de Touro (A Coruña, España)

Estos objetivos principales se dividen a su vez en objetivos más específicos:

- Evaluar cómo afectan estos tratamientos en las diferentes profundidades estudiadas

- Evaluar el efecto de los tratamientos sobre las concentraciones fitodisponibles de metales.

Justificación y objetivos

- Evaluar el efecto de los tratamientos sobre factores clave pH, carbono, nitrógeno, capacidad de intercambio catiónica y nutrientes.

- Evaluar el papel de Brassica juncea L. en el efecto de enmiendas aplicadas. Conocer qué tipo de función fitoremediadora cumple cultivada en estos tratamientos y en este tipo de suelo.

88 Justificación y objetivos

Justificación y objetivos

88 Justificación y objetivos

Justificación y objetivos

4. Capítulo 1. Changes in phytoavailable concentrations in a mine soil following the application of technosols and biochar with Brasica juncea L.

Justificación y objetivos

CHANGES IN PHYTOAVAILABLE CONCENTRATIONS IN A MINE SOIL FOLLOWING THE APPLICATION OF TECHNOSOLS AND BIOCHAR WITH BRASSICA JUNCEA L.

Rubén Forján1,*, Alfonso Rodríguez-Vila1, Rafael Silva Guedes2 and Emma F. Covelo1 1Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, Lagoas, Marcosende, Vigo, Pontevedra, Spain 2Institute of Agricultural Sciences, Federal Rural University of Amazonia, Belém, Brazil

ABSTRACT

Mining activities to obtain minerals cause soil to become contaminated and degraded. New techniques involving technosols and biochar are starting to be used with the aim of recovering these soils, together with phytoremediation, which is a cheap, ecological method. In this experiment, these two techniques were combined to create different treatments combining technosol, biochar and Brassica juncea L. For this reason, one of the objectives of this study was to determine the capacity

* Corresponding Author E-mail: [email protected].

93 2 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al.

of biochar to fix metals and enhance the positive effects of technosols. Another objective was to find out how Brassica juncea L. can enhance the effect of treatments using technosol and biochar. The greenhouse experiment was carried out in cylinders with the mine soil and the different treatments. The effects of the treatments were studied at three different heights over the 45-centimetre length of each cylinder. The study lasted a total of 11 months, using a settling pond from a depleted copper mine in Touro (Galicia, north-west Spain). The results of this experiment revealed that the phytoavailable concentrations of Cu, Pb, Ni and Zn decreased the most in the settling pond soil to which the treatments using technosol and biochar were applied. Brassica juncea L. proved to be capable of phytostabilising Cu, Pb, Ni and Zn, especially in the treatment using technosol and biochar. In turn, Brassica juncea L. could be a good translocating species for Pb and Zn, as it is capable of accumulating both metals in a satisfactory way.

1. INTRODUCTION

Mineral resources are a key element for socioeconomic progress. However, mining activities are the major sources of metal contamination as they cause a huge amount of potentially toxic elements to be released into the environment, particularly in terms of metal contamination, through runoff erosion caused by wind and water, leading to them being dispersed over wide areas in their bioavailable forms (Nouri and Haddioui 2016; Qasim 2015). One typical problem is acid mine drainage which contains very low pH and high metal content. Pollutants in soils are not only harmful to ecosystems and agricultural production, but also a serious threat to human welfare. For example, it has been estimated that 3.5 million locations in industrial and mining sites, landfills, energy production plants, and agricultural land are potentially contaminated in Europe, and as a result, soil contamination has been identified as an important issue for action in the European Community strategy for soil protection (Zhang et al. 2013). Remediation strategies that provide in situ immobilisation of contaminants, rather than the ex situ removal and dumping of soils, are generally more cost-effective and environmentally friendly. In addition, as well as diluting pollutants, these materials can reduce the mobility of metals due to various mechanisms, such as adsorption, precipitation, or complexation, which decrease the (bio)availability of pollutants in soils (Puga et al. 2015). For decades, one of these strategies has been the application of technosols due to their multifunctionality, as they improve soil quality (Asensio et al. 2013),

94 Changes in Phytoavailable Concentrations in a Mine Soil Following … 3 recycle waste and are able to fix metals. Technosols are a new group of soils that are strongly influenced by human technical activity. Furthermore, they can be made from waste and used in the subsequent regeneration of degraded or polluted soils. As a result, these materials are no longer considered as waste, and a value-added product is generated (Macía et al. 2014). One disadvantage of technosols is that some of their source materials can contribute certain amounts of metals to the soil (Yao et al. 2009). On the other hand, a recently applied strategy for recovering soils contaminated by metals is the use of biochar as a soil amendment. Biochar is the solid product left after the pyrolysis of waste biomass residues. Typically, most of the carbon in biochar has an aromatic structure that is highly recalcitrant in the environmental, a high pH value and cation exchange capacity, and is capable of enhancing soil productivity (Zhang et al. 2013). When used as a soil amendment, biochar often has a liming effect, with a microporous structure that results in a high specific surface area and active functional groups on the surface, which implies a high capacity for complex metals on their surface. The surface sorption of metals on biochar has been demonstrated on multiple occasions using scanning electron microscopy (Beesley and Marmiroli 2011; Lu et al. 2012). If biochar is applied to soil, the negative charge and therefore the cation exchange capacity (CEC) may increase due to the induced higher pH, and in turn, the electrostatic attraction between metal cations and soil particles will become stronger. Biochar can stabilize metals in contaminated soils, improve the quality of the contaminated soil, and significantly reduces the uptake of metals by crops. Therefore, the application of biochar can potentially provide a new solution for remediating soils contaminated by metals, and can enhance soil productivity (Karer et al. 2015, Paz-Ferreiro et al. 2014, Zhang et al. 2013). The reduction in the bioavailability of metals and other modifications to the substrate induced by the application of biochar may be beneficial to establishing plant cover on top of waste, to achieve long-term phytostabilisation (Puga et al. 2015). Pilon-Smits (2005) interestingly defined phytoremediation as ‘the use of plants and their associated microbes for environmental cleanup’, which is a cost-effective, non-invasive alternative technology for engineering-based remediation methods. One type of phytoremediation is phytoestabilisation. Phytostabilisation is a less invasive, low-cost phytotechnology, used on its own or in combination with amendments as a potential option to restore the physical, chemical, and biological properties of potentially toxic trace elements in contaminated soils (Concas et al., 2015, Hattab et al., 2014). Also, phytoextraction is a phenomenon in which hyperaccumulator plants absorb

95 4 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al. metals from soil through the root system and translocate them to the harvestable shoot, making it possible to recover metals from the harvestable parts of plants. Phytoextraction is based on the mechanism of hyperaccumulation, while phytostabilisation is based on the mechanism of surface complexation, and is involved in the phenomena of metal sorption (Mani and Kumar 2014). As most experiments have been carried out with biochar pots or small quantities in the laboratory and studied in top soils (0–25 cm) (Hattab et al. 2014), in this study we decided to scale up these experiments and extend to a depth of 45 cm. Another aim of this study was to determine the capacity of biochar to fix metals and enhance the positive effects of technosols when supplemented with Brassica juncea L. plants. Brassica juncea L. has been used successfully for the phytoextraction of metals from polluted mine soils (Lombi et al. 1999; do Nascimento et al. 2006; Rodríguez-Vila et al. 2014). Cu, Pb, Ni, and Zn were chosen for this experiment, as metals such as Cu, Pb and Zn are particularly important since high quantities of these metals can reduce crop production due to the risk of biomagnifications and bioaccumulation in the food chain (Rahman et al. 2013). A further objective was to discover how Brassica juncea L. can strengthen and enhance the effect of treatments using two different amendments (technosol and biochar). With this aim in mind, in this study we inserted 45-cm long cylinders into the settling pond soil from a copper mine, in order to reflect as closely as possible the first few centimetres of the settling pond in the field. The settling pond soil had been treated with different combinations of technosol and biochar, and Brassica juncea L. had been planted. The effect of the treatments was studied at three different heights over the 45-centimetre length. The study lasted 11 months, and the settling pond is in the depleted copper mine located in Touro (Galicia, north-west Spain).

2. MATERIAL AND METHODS

2.1. Soil Sampling

The sample zone was located in an old copper mine in Touro (Figure 1), north western Spain (8º 20' 12.06'' W 42º 52' 46.18'' N). The climate in this zone is Atlantic (oceanic) with precipitation reaching 1886 mm per year (with an average of 157 mm per month) and a mean daily temperature of 12.6ºC. The average relative humidity is 77% (AEMET, 2015). In order to carry out

96 Changes in Phytoavailable Concentrations in a Mine Soil Following … 5 the study, one soil and three amendments were selected. The chosen soil came from the settling pond (S) at the Touro mine, and the three amendments were sand (SS) provided by the company Leboriz S.L.U., used as a neutral control; technosol (T) provided by the company Tratamientos Ecológicos del Noroeste (T.E.N.) and biochar (B) provided by the company Proininso S.A.

Figure 1. Location of the sampled area in Touro mine.

The settling pond soil (S) was comprised of waste material resulting from the flotation of sulphides during copper processing. The pool has been dry for several years, and is considered to be soil according to the latest version of the FAO (2006). The sand (SS) consisted of washed sea sand. The technosol (T) consisted of a mixture of 60% purified sludge (from a waste water treatment plant), 10% sludge from an aluminium company (Padrón, La Coruña, Spain) 5% ash (Ence, a cellulose production plant in Pontevedra, Spain), 10% waste from the agri-food industry (canning plants and Ecogal), 5% sands from purification plants (sand fraction), plus a further 10% of materials whose contents are not precisely known due to the privacy policy of the company. The biochar (B) used was made from Quercus ilex wood with a pyrolysis temperature of 400°C for 8 h.

97 6 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al.

2.2. Greenhouse Experiment

 Settling pond soil + technosol (ST)  Settling pond soil + Technosol + vegetated with Brassica juncea L. (STP)  Settling pond soil + Technosol + biochar (STB)  Settling pond soil + Technosol+biochar+ vegetated with Brassica juncea L. (STBP).

The amendment ratios used are detailed in Table 1. The total weight of each cylinder was 3.5 kg. The experiment was carried out over 11 months at a controlled temperature and humidity (temperature of 22±2°C, and 65±5% relative air humidity). A total of 90 cylinders (15 cylinders per treatment) were prepared and distributed at random (S, SS, ST, STP, STB, STBP) (Figure 2). Three cylinders of each type were withdrawn at 3 different times: Time 1= 3 months, Time 2= 7 months, Time 3= 11 months. The meshes were removed from the cylinders and processed for analysis at 3 different heights: the first from 0-15 cm, the second from 15-30 cm, and the third from 30-45 cm (Figure 2). The cylinders were watered to field capacity throughout the experiment.

Table 1. Proportions used to make the controls and the different treatments

Soil Sand Technosols Biochar S 100% SS 85% 15% ST 85% 15% STP 85% 15% STB 85% 11% 4% STBP 85% 11% 4%

98 Changes in Phytoavailable Concentrations in a Mine Soil Following … 7

Figure 2. Cylinder design and the different heights.

2.3. Soil Analysis

99 8 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al.

CN-2000 module using solid samples. Exchangeable cations were extracted with 0.1 M BaCl2 (Hendershot and Duquett 1986) and their concentrations determined by ICP-OES (Optima 4300 DV; Perkin-Elmer). Phytoavailable content of copper, nickel, lead and zinc was extracted with 0.01 M CaCl2 in soil solution (Houba et al. 2000). Pseudototaltotal metal contents were extracted with aqua regia by acid digestion in a microwave oven (Milestone ETHOS 1, Italy). Metal concentrations were determined by ICP-AES (Optima 4300 DV; Perkin-Elmer). Pseudototal concentrations were compared with the generic reference level (GRL) established for Galician soils (Macías and Calvo de Anta 2009).

2.4. Plant Growth and Determination of Metals in Plant Tissues

The Brassica juncea L. were pre-germinated in seedbeds until they grew two fully expanded leaves, were transferred to the cylinders (STP, STBP). The plants were harvested in the same state of maturity, for comparison in the same state of development at the three periods (Time 1= 3 months, Time 2= 7 months, Time 3= 11 months). Growth was allowed under greenhouse- controlled conditions, with a photoperiod of 11:13 light/dark, temperature of 22±2°C and 65±5% relative air humidity. At the end of each period, the roots and shoots were divided and carefully washed with deionised water. Fresh biomass was weighed immediately, and dry mass was assessed after oven drying for 48 h at 80°C and cooling at room temperature. The plant tissues, divided into roots and shoots, were air dried and ground up. The total concentrations of Cu, Ni, Pb and Zn in the Brassica juncea L. were extracted by acid digestion using a mixture of H2O2 and HNO3 (1:6v/v) in a microwave oven (Milestone ETHOS 1). In order to establish the phytoremediation capacity of Brassica Juncea L., different factors were calculated in this experiment, the transfer coefficient (TrC) and the translocation factor (TF). Using these factors, the pseudototal concentrations were related to the Brassica Juncea L. contents. The transfer coefficient (TrC) in the plants that were studied measured their efficiency in taking up metals from the soil, and was calculated using the following equation:

TrC= Cp/Cso

100 Changes in Phytoavailable Concentrations in a Mine Soil Following … 9 where TrC represents the transfer coefficient of the plants, Cp is the metal concentration in the shoots (mg kg-1) and Cso is the metal content of the soil (mg kg-1) (Karami et al. 2011; Peijnenburg and Jager 2003). A plant is considered to be an accumulator or hyperaccumulator biosystem when TC > 1 (Busuioc et al. 2011). The translocation factor (TF) for the plants is expressed by the following equation:

TF= Cs/Cr where Cs and Cr are the metal concentrations (mg kg-1) in shoots and roots, respectively. A TF with a high value indicates a relatively high shoot metal concentration compared to its root concentration. A plant species translocates metals effectively from the roots to shoots when TF > 1 (Baker and Brooks 1989).

2.5. Statistical Analysis

All of the analytical determinations were performed in triplicate. The data obtained were statistically treated using the programme SPSS version 19.0 for Windows. Analysis of variance (ANOVA) and a test of homogeneity of variance were carried out. In case of homogeneity, a post hoc least significant difference (LSD) test was carried out. If there was no homogeneity, Dunnett’s T3 test was performed. Student’s t test was used to compare the results of TrC and TF between STP and STBP.

3. RESULTS

3.1. General Characteristics of the Settling Pond Soil (S), Sand (SS), Technosol (T), and Biochar (B)

The soil from the settling pond (S) and the neutral control (sand, SS) had an acidic pH, while the technosol and biochar (T and B) had higher pH values (P < 0.05) (Table 2). The biochar had the highest pH (Table 2). Total carbon (TC) was significantly higher in the biochar and technosol (T) compared to the soil from the settling pond and sand (P < 0.05) (Table 2). The technosol had the highest total nitrogen content (TN) (P < 0.05) (Table 2). TN was extremely

101 10 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al. low in the soil and in the sand; in fact, in the soil it was undetectable (Table 2). The exchangeable cation capacity (CEC) of T and B was significantly higher than in the controls (S and SS). The pseudototal concentration of Cu in the settling pond soil was higher than in the amendments used (T and B) (P < 0.05) (Table 2). The technosol had the highest pseudototal concentration of Pb and Zn, especially Zn (table 2). The biochar and technosol had higher pseudototal concentration of Ni than S and SS (Table 2). S and T had pseudototal Cu and Zn concentrations that were higher than the GRL for Galician soils (Table 2), while the pseudototal Pb concentrations in T exceed the GRL (Table 2). The extractable CaCl2 concentration of Cu, Pb and Ni in S was significantly higher than in the T and B (P < 0.05) (Table 2). The CaCl2- extractable concentration of Zn in T was higher than in S, SS and B (Table 2). The biochar had no detectable CaCl2-extractable concentrations of Cu and Pb (Table 2).

Table 2. Selected characteristics and metal concentrations of settling pond soil (S), sand (SS), technosoil (T) and biochar (B)

S SS T B GRL pH 2.73±0.07d 3.83±0.55c 6.04±0.05b 9.90±0.02a Total (g/kg) 1.93±0.15c 2.76±0.60c 256±2.51b 676±4.58a Carbon Total (mg/kg) u.d 0.10±0.01c 17.6±0.50a 5.34±0.22b Nitrogen -1 CEC (cmol(+)kg ) 6,11±0,05c 0,14±0,01d 76,6±4,80a 15,8±17,8b Cu 637±2.08a 46.4±1.14c 226±5.13b 27.1±1.24d 50 Pb Pseudototal 16.1±1.00b 10.4±0.56c 89.6±1.52a u.d 80 Ni (mg/kg) 16.4±1.10b 8.26±1.05c 26.3±0.57a 25.0±2.00a 75 Zn 65.4±2.51b 18.9±1.20c 340±5.50a 62.6±1.95b 200 Cu 139±2.08a 3.11±0.17b 6.01±0.03b u.d Pb 0.65±0.03a 0.11±0.01c 0.33±0.02b u.d Ni CaCl2 2.25±0.30a 0.05c 1.03±0.02b 0.33±0.02c Zn (mg/kg) 64.4±1.24b 0.32±0.01c 165±1.63a 1.24±0.01c For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level. Typical deviation is represented by ±. GRL= generic reference level (GRL) established for Galician soils (Macías and Calvo de Anta 2009).

102 Changes in Phytoavailable Concentrations in a Mine Soil Following … 11

3.2. Evolution of the Pseudototal Concentrations of Cu, Pb, Ni, Zn at Three Heights and over the 11-Month Period

In general, the ST treatment (settling pond soil treated with technosol) had the highest Pb concentration over time and at different heights (Figure 3) The pseudototal concentration of Ni was higher in treatments ST, STP, STB and STBP than in controls S and SS in all cases except at time1- height 3 (P < 0.05) (Figure 3b). The STB and STBP treatments had significantly higher pseudototal Zn concentrations than controls S and SS at height 1 over time (Figure 3a, 3b). At height 2, ST had the highest zinc content at times 1-3 (Figure 3c, 3d). Finally, at height 3, the SS had the highest pseudototal Zn concentration over time (P < 0.05) (Figure 3e, 3f). At heights 1 and 2, at time 1-3 the settling pond soil (S) had a higher pseudototal concentration of Cu than the neutral control (SS) and the settling pond with different treatments (Figure 3a,3b, 3c, 3d). Overal, the treated settling pond had significantly higher pseudototal concentrations of Pb, Ni, Zn than the control treatments (S and SS) (P < 0.05) (Figure 3). At height 3, S and SS had the highest pseudototal concentrations of Cu. The settling pond soil that was only treated with technosol (ST) had the highest pseudototal concentration of Pb at time 1-3. The SS had the highest pseudotal Zn concentration at time 1-3 (Figure 3e, 3f). In the case of Ni, at time 1 the highest was in SS, but at time 3 the highest was in STP and STBP (P < 0.05) (Figure 3e,3f).

Figure 3. (Continued).

103 12 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al.

104 Changes in Phytoavailable Concentrations in a Mine Soil Following … 13

Figure 3. Evolution of the pseudototal concentrations of Cu, Pb, Ni, and Zn at three heights and over the 11-month period. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represent standard deviation.

3.3. Evolution of CaCl2-Extractable (Phytoavailable) Contents of Cu, Pb, Ni, Zn at Three Heights and over the 11-Month Period

At height 1, the settling pond soil (S) had a higher CaCl2-extractable Cu, Pb, and Ni concentration, at the three times, than in the treated settling pond

105 14 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al. soil; the STP treatment had a significantly equal content to S at time 1 (P < 0.05) (Table 3.1). STBP had the lowest values of CaCl2-extractable concentrations for all metals (Table 3.1). At height 2, S and SS had the highest CaCl2-extractable Cu at time 1. STP had the highest CaCl2-extractable concentrations of Pb, Ni and Zn at time 1-2, except for Zn, which was the highest in STB at time 1(P < 0.05) (Table 3.2). At time 3, the controls (S, SS) had the highest CaCl2-extractable concentrations of Cu and Ni, and the settling pond soil with different treatments had the highest CaCl2- extractable concentrations of Pb and Zn (P < 0.05) (Table 3.2). At height 3, S at time 1-2 had the highest CaCl2-extractable concentration of Pb (Table 3.3). The neutral control and the treated settling pond had higher CaCl2-extractable concentrations of Cu, Ni and Zn than in S at time 1 and 2 (P < 0.05) (Table 3.3). At time 3, the S and SS were overall those with the highest CaCl2- extractable concentrations of all metals (Table 3.3).

Table 3.1. Evolution of CaCl2-extractable (Phytoavailable) concentrations Cu, Pb, Ni, Zn (mg.kg-1) at height 1 and over the 11-month period

Height S SS ST STP STB STBP 1 Cu 50.6±3.25a 3.37±0.07d 6.06±0.10c 6.64±0.09b 2.09±0.14f 2.54±0.10e

Pb 0.41±0.08a 0.15±0.01e 0.39±0.04b 0.41±0.05a 0.23±0.01d 0.30±0.02c Ni 2.06±0.05a 0.07±0.01f 1.03±0.32c 0.22±0.01d 0.19±0.6e 1.54±0.70b

Time 1 Time Zn 3.09±0.87e 0.42±0.02e 166±6.66a 160±1.47b 151±6.22c 145±3.54d Cu 54.5±6.82a 5.30±0.20b 7.27±.091b 7.61±0.73b 1.16±1.00c 1.77±0.06c

Pb 0.44±0.02a 0.15±0.09d 0.37±0.02b 0.37±0.05b 0.22±0.01c 0.21±0.01c Ni 2.53±0.41a 0.02e 0.84±0.07c 0.94±0.10c 0.71±0.03d 1.55±0.29b

Time 2 Time Zn 2.63±0.09d 0.59±0.88d 157±4.01b 166±1.98a 155±4.21b 134±3.69c Cu 54.1±3.13a 4.76±0.39b 4.49±0.51b 4.11±0.07b 1.13±0.14c 1.22±1.00c

Pb 0.44±0.04a 0.19±0.21e 0.38±0.01b 0.35±0.02c 0.30±0.01d 0.35±0.02c Ni 3.32±0.25a 0.02e 1.28±0.06b 1.07±0.04c 1.03±0.02c 0.93±0.01d

Time 3 Time Zn 2.34±0.70d 0.31±0.06d 167±0.75b 175±3.11a 165±1.39b 151±4.02c For each row. different letters in different samples means significant differences (n=3. ANOVA; P<0.05). Typical deviation is represented by ±.

106 Changes in Phytoavailable Concentrations in a Mine Soil Following … 15

Table 3.2. Evolution of CaCl2-extractable (Phytoavailable) concentrations Cu. Pb. Ni. Zn (mg.kg-1) at height 2 and over the 11-month period

Height S SS ST STP STB STBP 2 Cu 50.9±1.31a 50.2±0.09a 48.7±0.98b 44.8±0.07c 42.3±0.11d 41.9±1.00e

Pb 0.40b 0.38±0.02c 0.41±0.01b 0.54±0.04a 0.32±0.02b 0.41±0.02b Ni 2.06±0.07f 2.09±0.09e 2.74±0.10d 3.62±0.22a 2.90±0.02c 3.21±0.17b

Time 1 Time Zn 2.37±0.06e 2.33±0.08e 4.65±0.05c 6.65±0.37a 5.91±0.17b 4.50±0.06d Cu 55.4±0.33b 53.3±0.01c 55.2±0.28b 56.5±0.51a 47.2±0.99d 44.0±1.68e

Pb 0.48±0.01ab 0.35±0.45e 0.46c 0.51±0.02a 0.43±0.01d 0.44±0.02d Ni 2.40±0.01d 2.38±0.01e 2.88±0.05b 3.21±1.84a 2.64±0.10c 2.88±0.07b

Time 2 Time Zn 2.59±0.01f 2.66±0.04e 5.29±1.02c 5.57±0.03b 5.69±0.05a 3.23±0.20d Cu 55.9±0.03b 57.2±0.06a 55.0±0.09b 55.5±0.04b 50.5±c 48.1±1.01d

Pb 0.43±0.01c 0.37±0.04d 0.47b 0.48±0.01b 0.47±0.01b 0.50±0.01a Ni 3.31±0.12a 2.48±0.06d 3.02±0.07b 3.00±0.03b 2.17±0.10e 2.74±0.05c

Time 3 Time Zn 2.58±0.17c 2.64±0.09c 5.59±0.49a 4.51±0.38ab 4.15±0.20b 3.62±0.41b For each row. different letters in different samples means significant differences (n=3. ANOVA; P<0.05). Typical deviation is represented by ±.

Table 3.3. Evolution of CaCl2-extractable (Phytoavailable) concentrations Cu, Pb, Ni, Zn (mg.kg-1) at height 3 and over the 11-month period

Height S SS ST STP STB STBP 3 Cu 52.0±0.01b 53.6±0.03a 52.8±0.02ab 51.4±0.03c 47.6±0.5e 50.1±0.01d

Pb 0.55±0.02a 0.43±0.01b 0.39±0.02c 0.40±0.01c 0.38d 0.32±0.03e Ni 2.10±0.05b 2.21±0.01b 2.92±0.02a 3.10±0.04a 3.00±0.03a 3.00±0.02a

Time 1 Time Zn 3.45±0.02e 2.55±0.01f 3.97±0.01c 4.26±0.05a 4.09±0.03b 3.72±0.01d Cu 53.1±1.76b 52.7±1.01b 59.0±3.61a 58.3±2.99a 50.4±1.07c 50.2±1.64c

Pb 0.55±0.49a 0.42±0.01c 0.43±0.02bc 0.45±0.02b 0.37±0.31d 0.37±0.29d Ni 2.57±1.02e 2.82±0.87c 3.30±2.64a 3.29±1.69a 2.96±0.04b 2.75±0.79d

Time 2 Time Zn 3.43±0.03d 3.00±1.99e 3.93±0.02b 3.02±2.22e 4.03±0.05a 3.55±0.50c Cu 58.3±1.11b 61.8±0.73a 58.1±0.89b 57.4±1.45bc 46.7±0.99c 46.3±0.83c

Pb 0.47±0.01a 0.49±0.02a 0.44±0.01b 0.45b 0.34±0.05c 0.43±0.02b Ni 3.48±0.09e 3.14±0.12c 3.01±0.04c 3.34±0.06b 2.85±0.06d 2.67±0.10e

Time 3 Time Zn 3.73±0.24a 3.02±0.01d 3.15±0.11b 3.04±0.01cd 3.10±0.04b 3.06±0.02c For each row. different letters in different samples means significant differences (n=3. ANOVA; P<0.05). Typical deviation is represented by ±.

107 16 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al.

3.4. Harvestable Amounts of Cu, Pb, Ni, Zn and Determination of Metals in Plant Tissues: Translocation Factor (TF) and Transfer Coefficient (TC)

Harvestable Amounts of Cu, Pb, Ni, Zn in Brassica Juncea The Brassica juncea L. was not capable of growing in the settling pond soil (S) or in the neutral control (SS), and for this reason is not shown in Figure 4. In the first harvest (Time 1), the concentrations of Cu, Pb, Ni and Zn in the Brassica juncea L. shoots and roots from the settling pond soil treated with technosol+biochar (STBP) were significantly higher than in the settling pond soil that was only treated with technosol (STP) (Figure 4a,4b; p < 0.05). In the second harvest (Time 2), STP had higher values of Cu, Pb and Zn concentrations in shoots than STPB. However, the metal concentrations in the roots did not reveal any significant differences between the different treatments (Figure 4c, 4d; p < 0.05). In the final harvest, the STBP had the highest values for all metal concentrations in shoots and roots (Figure 4e, 4f). Translocation Factor (TF) The TF at time 1 for all metals (Cu, Pb, Ni, and Zn) was the highest in the settling pond soil that was only treated with technosol (STP) (Figure 5a). At time 2, STPB 2 had a higher TF for Cu, Ni and Zn than in STP, and the highest Pb values were found in STP (P < 0.05) (Figure 5b). The TF at time 3 for Cu did not reveal any differences between the different treatments, although the TF for Pb and Zn was significantly higher in STBP than in STP; in the case of the TF for Ni, STP had the highest values (Figure 5c).

108 Changes in Phytoavailable Concentrations in a Mine Soil Following … 17

Figure 4. (Continued).

109 18 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al.

Figure 4. Harvestable amounts of Cu, Pb, Ni, Zn and determination of metals in plant tissues. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represent standard deviation.

110 Changes in Phytoavailable Concentrations in a Mine Soil Following … 19

Figure 5. Translocation factor (TF) of Cu, Ni, Pb, and Zn to and within mustards according Translocation factor (TF) (shoots concentration/roots concentration) over the 11-month period. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represent standard deviation.

111 20 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al.

Transfer Coefficient (TrC) At time 1, the TrC for Cu, Pb and Zn was significantly higher in STP than in STBP, but STP had the highest TrC for Ni (Figure 6a). At time 2, STBP had the highest TC for Ni and Zn. STP had the highest values of TrC for Pb. The TrC for Cu did not reveal any significant differences between the different treatments (P < 0.05) (Figure 6b). The TrC for Cu at Time 3 did not reveal any significant differences between the different treatments. The values of TrC for Ni and Zn in STP were significantly higher than in STBP, and in the case of TrC for Pb, STBP had the highest values (Figure 6c).

112 Changes in Phytoavailable Concentrations in a Mine Soil Following … 21

Figure 6. Transfer of Cu, Ni, Pb and Zn to and within mustards according to transfer coefficient (TrC) (shoots concentration/pseudo-total soil concentration) over the 11- month period. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represent standard deviation.

4. DISCUSSION

4.1. Evolution of the Pseudototal Contents of Cu, Pb, Ni, Zn at Three Heights and Over the 11-Month Period

The high Cu content in S is due to its origin, as it was a soil from the settling pond in a copper mine, predominated by wastes resulting from the flotation of sulphides during copper processing. Once the different treatments were applied, the pseudototal content of Cu decreased, although the pseudototal content of Pb, Ni and Zn increased. This increase occurred at all heights, and is mainly due to the materials that were used to make the technosol, such as purification plant waste, waste from the agri-food industry or sands from purification plants (Amir et al. 2005; Smith 2009; Travar et al. 2015; Weber et al. 2007). The metal that was contributed the most by this technosol was Zn. This may be because some of the components used to make this technosol contain this metal, mainly the purification plant sludges (Rorat et al. 2016, Tai et al. 2016). This increase in the concentration of Zn due to the application of technosol only surpassed the GRL at height 1 (Macías and Calvo de Anta 2009). It should be noted that despite the increase in the pseudototal content of Pb, Ni and Zn in S due to the treatments, the

113 22 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al. phytoavailable percentage of concentrations with respect to the pseudototal decreases. The mean percentages of phytoavailable concentrations with respect to the pseudototal for soil S at height 1 were 9.6% Cu, 1.49% Pb, and 20.86% Ni, while the mean percentages for the treated soils were 1.48% Cu, 0.49% Pb, and 2.80% Ni. The only case when the percentage of phytoavailable concentrations increased with respect to the pseudototal was in Zn, with a value of 6.41% in S, and in the settling pond where we applied the treatments, with a mean value of 37.06%. This reduction in the percentage of phytoavailable concentrations with respect to the pseudototal of Cu, Pb and Ni is due to the capacity of the technosol and biochar to fix these metals, and is also possibly due to the contribution of organic matter and an increased pH caused by the treatments. Some authors, such as McBride et al. (1997) found linear relationships between the activity or solubility of several metals and the soil properties of pH, organic matter content, and pseudototal metal concentration.

4.2. Evolution of the CaCl2-Extractable (Phytoavailable) Concentrations of Cu, Pb, Ni, Zn at Three Heights and over the 11-Month Period

The effect of the different treatments is clearer at height 1, especially in the case of the treatments STB and STBP, which generally have the lowest values for phytoavailable concentrations of Cu, Pb and Ni. This is due to the positive effect of the biochar when it is combined with the technosol. Other authors such as Fowles (2007) have previously observed a greater positive effect on the soil properties by applying organic amendments that contained biochar amongst their components, compared with organic amendments that did not contain boichar. On the one hand, the positive effect of technosol is due to the elements from which it is made (solid urban waste, ash from a paper company, remnants from agri-food industries and sludge from a purification plant). Various authors have demonstrated that these wastes have a high organic matter content and high concentrations of basic cations; this high concentration of basic cations has a direct repercussion on the high CEC and pH (Amir et al. 2005; Weber et al. 2007). On the other hand, these positive features of the technosol were improved by the biochar, due to the fact that the biochar has a negative charge, and therefore the cation exchange capacity (CEC) may increase due to the induced higher pH, and in turn, the electrostatic attraction. Also, biochar has a large surface area and high organic matter

114 Changes in Phytoavailable Concentrations in a Mine Soil Following … 23 content (Karer et al. 2015; Puga et al. 2015, Beesley and Marmiroli 2011). As the technosol used has a mean phytoavailable Zn concentration of 165 (mg.kg- 1) and the fact that the settling pond soil had a very acidic pH, the treatments applied were not able to mitigate the increase in phytoavailable Zn. Also, the positive effect of the biochar was once again seen, as the STB and STBP treatments also had lower phytoavailable concentrations of Zn than ST and STP. At height 2, the effect of the treatments was not so clear. In fact, at times 1 and 2, the settling pond soil to which the treatments were applied generally had a higher phytoavailable concentration of the metals that were studied, especially in the case of STP. This increase in metals was due to them being provided by the treatments that were applied, through the materials used to make the amendments (Rorat et al. 2016, Smith 2009; Travar et al. 2015; Weber et al. 2007) (Table 4). The treatments at this height were not able to counteract the deficiencies in S, such as its low pH and organic matter content, which plays a crucial role in metal solubility and means that the metals are in a phytoavailable form in the soil (Park et al. 2011; Temminghoff et al. 1997; Weng et al. 2001). Amongst the different treatments that were applied, those that performed the best throughout the experiment were once again those that combined technosol with biochar (STB, STBP). There is one exception in the case of Pb in STBP at time 3; this increase in the concentration of phytoavailable Pb may be due to the fact that the increase in pH and organic matter at this height was not enough to immobilise this element. At height 3, the positive effect of the treatments, especially STB and STBP, in the distribution of phytoavailable concentrations in the settling pond soil, followed the same pattern of behaviour for all of the metals that were studied. It was observed that in times 1 and 2, the settling pond soil to which the treatments were applied had higher concentrations of all of the metals except for Pb, in comparison with the controls (P<0.05). On reaching time 3, the phytoavailable concentrations of the studied metals were lower in the settling pond soil to which the different treatments were applied, once again especially in STB and STBP (P<0.05). This reduction in the phytoavailable concentrations may be due to the increase in the pH, TC and CEC caused by the treatments, as the amendments used to make these treatments had a high pH, CEC and high TC concentration (T and B, Table 2). These increased in TC, pH and CEC have a positive effect on the reduction of the phytoavailable concentrations, as has been demonstrated by various authors (Park et al. 2011; Temminghoff et al. 1997; Weng et al. 2001).

115 24 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al.

4.3. Uptake and Transfer of Metals to Mustards

Harvestable Amounts of Cu, Pb, Ni, Zn In the Brassica juncea L. from the STBP treatment, a trend was observed during the experiment whereby the harvestable amounts of Cu, Pb, Ni and Zn in the roots and shoots were higher than in the STP treatment. Zn is the metal with the lowest affinity for the amendments used to make the STBP treatment, and so the phytoavailable concentrations of Zn would be more available for the plants than, for example, those of Cu and Pb (Forján et al. 2016). This would explain why, in nearly all of the cases, the plants had a higher concentration of Zn than the other metals. These higher harvestable amounts of Cu, Pb, Ni and Zn in the plants from the STBP treatment in comparison with those from the STP treatment may be due to the positive effect of the biochar on the soil, which makes the plants develop more, with the ability to absorb the metals more effectively. Biochar is a soil conditioner that enhances plant growth by supplying and retaining nutrients, and by providing other benefits such as improving the physical, chemical and biological properties of the soil (Fellet 2011). As previously mentioned, the STBP treatment had a positive effect on S, providing significantly lower concentrations of phytoavailable Cu, Pb and Ni; in addition to this positive effect is the fact that the plants grown in STBP were those with the highest content of these metals. This shows the positive effect of combining technosol+Biochar + Brassica juncea L. on the metals that were studies (Cu, Pb, Ni, and Zn), confirming what other authors have demonstrated over the last few years: that the application of biochar in metal-contaminated soil for improving plant growth can also lead to an increase in metal uptake by some plants (Prapagdee et al. 2014).

Transfer Coefficient (TrC) The Brassica juncea L. plants grown in the settling pond soil to which the different treatments were applied (STB, STBP) would not have a phytoextracting capacity for the metals that were studied, as these plants did not have TrC values higher than 1 in any of the treatments, taken as the base value from which a plant is considered as an accumulator or hyperaccumulator biosystem (Busuioc et al. 2011). Also, these low TrC values indicate that Brassica juncea L. could act as a phytostabilising plant for Cu, Pb, Ni and Zn, as the ideal plant species for phytostabilisation are the so-called metal excluders, which have a low root to shoot transfer coefficient (Kidd et al. 2009). This phytostabilsing capacity of Brassica juncea L.is important, as the

116 Changes in Phytoavailable Concentrations in a Mine Soil Following … 25 phytostabilisation technique is the most suitable for relatively immobile materials and large surface areas, and is currently acceptable for the remediation of mining sites (Cunningham et al. 1995). This means that the exposure of livestock, wildlife and human beings to these metals is reduced (Mani and Kumar 2014).

Translocation Factor (TF) During the experiment, the TF values evolved from being higher in the plants harvested from the STP treatment, to being higher in those harvested from the STBP treatment, possibly due to the capacity of the Biochar to retain nutrients, leading to the improved development of the plants. In the STBP treatment, the TF values for Pb and Zn gradually rose during the experiment, and at time 3 even exceeded the value of 1. According to Baker and Brooks (1989) a value of TF>1 means that the plant is capable of translocating metals effectively from the roots to shoots. This increase in the value of TF at time 3 suggests that Brassica juncea L. may be a good translocator species for Pb and Zn once the treatment has become stabilised, and that this species may accumulate Pb and Zn in a satisfactory way without reaching the point of being a hyperaccumulator (Rotkittikhun et al. 2007). Salido et al. (2003) demonstrated the effectiveness of Brassica juncea L. to phytoremediate soil contaminated by lead. As regards the Cu and Ni, the fact that TF<1 does not mean that the plant is not an effective phytoremediator, but that instead of translocating these metals from the root to the shoot, it fixes them in the root (Nouri et al. 2009). This behaviour with Cu and Ni confirms, as in the case of the TC, that Brassica juncea L. grown in the STBP treatment can be a good phytostabilising species for these metals.

CONCLUSION

The settling pond soil to which the treatments made of technosol and biochar were applied (STB and STBP) were those that had the best result in terms of reducing the phytoavailable content of Cu, Pb, Ni and Zn. This shows the positive effect of biochar, which increases the capacity of the technosol to reduce these phytoavailable concentrations. The phytoavailable concentrations of Cu, Pb and Ni were considerably reduced at heights 1 and 3 as the treatments were stabilised. At height 2, the treatments that were applied were able to reduce the phytoavailable concentrations of Cu and Ni, but not of Pb or Zn. This shows that the treatments STB and STBP, albeit with certain minor

117 26 Rubén Forján, Alfonso Rodríguez-Vila, Rafael Silva Guedes et al. exceptions, are effective when reducing the concentrations of phytoavailable metals. Brassica juncea L. proved to be capable of phytostabilising Cu, Pb, Ni and Zn, especially in the treatment made of technosol and biochar. Brassica juncea L. also proved that it can be a good translocator species for Pb and Zn once the treatment has stabilised, and can accumulate Pb and Zn in a satisfactory way without reaching the point where it can be considered as a phytoextractor. This means that according to the values of TrC and TF, Brassica juncea L. combines a capacity as a phytostabiliser and accumulator when grown on these treatments, thereby completing the ability of these treatments to fix Cu, Pb, Ni and Zn. It can therefore be concluded that the combination of technosol with biochar and Brassica juncea L. makes an effective treatment when reducing the phytoavailable concentrations of Cu, Pb, Ni and Zn in settling pond soil.

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Capítulo 2. Application of Compost and Biochar with Brassica juncea L. to Reduce Phytoavailable Concentrations in a Settling Pond Mine Soil

Waste Biomass Valor DOI 10.1007/s12649-017-9843-y

ORIGINAL PAPER

Application of Compost and Biochar with Brassica juncea L. to Reduce Phytoavailable Concentrations in a Settling Pond Mine Soil

Rubén Forján1 · Alfonso Rodríguez‑Vila1 · Nuria Pedrol1 · Emma F. Covelo1

Received: 28 November 2016 / Accepted: 28 January 2017 © Springer Science+Business Media Dordrecht 2017

Abstract After they are closed, mines impact the envi- Keywords Biochar · Metal · Settling pond mine soil · ronment by contaminating air, water, soil, and wetland Phytostabilisation · Compost sediments from the scattered tailings, and by polluting the groundwater with discharged leachate. Touro mine is depleted copper mine (in Galicia, north-west Spain), the Introduction settling pond soil has high bioavailable metal concentrations (mainly high Cu concentrations), extreme pH values, Mineral resources are a basic cornerstone of socioeconomic a low cation exchange capacity and organic matter. This progress. However, in the absence of remediation, after study aimed to determine the capacity of biochar to fix met- mines are closed they impact the environment by contami- als (particularly in the reduction of Cu concentrations) and nating the air, water, soil, and wetland sediments from the enhance the positive effects of compost, supported by the scattered tailings, as well as polluting groundwater by dis- phytoremediation capacity of Brassica juncea L. In this charged leachate. The persistent erosion of mine soils can experiment, brassica was chosen because several authors affect land stability, revegetation efforts, and water quality. used this plant satisfactorily for similar purposes. The Thus, mining causes substantial damage to the environment greenhouse experiment was carried out in 45-cm cylinders, worldwide, despite being an important economic activity. and the effects of the treatments were studied at different Today, mining restoration is mandatory in most countries soil depths. The study lasted a total of 11 months and was [1–3]. carried out in the settling pond of Touro mine. At depth Different technologies have been developed to remove 0–15 cm, the treatments applied exhibited the best effect on metal(loid)s from soils. Some of them make use of chemi- the reduction of the phytoavailable metal concentrations. cal precipitation, oxidation–reduction, ion exchange, elec- At depth 15–30 cm, the treatments showed better results at trochemical procedures, and filtration. However, these Time 2 than at Times 1 and 3. Only at depth 0–45 cm and methods may have significant disadvantages, such as at Time 3, a better behaviour of the treatment elaborated incomplete extraction of metals, the need for special equip- with compost + biochar + B. juncea L was observed. The ment, and sometimes high and costly energy requirements TC and TF values revealed that the cultivated B. juncea L. [4]. Remediation strategies that provide the in situ immo- presented good phytostabilising capacity for Cu, Pb, Ni, bilisation of contaminants, such as amendments made and Zn. of waste materials, are generally more cost-effective and many times environmentally friendly. In addition to diluting pollutants, these materials can also reduce the mobility of metals by a series of mechanisms, such as adsorption, * Rubén Forján precipitation, or complexation, which decrease the (bio) [email protected] availability of pollutants in soils [5]. Apart from their positive effect on recovering the soil, the use of these amend- 1 Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, Lagoas, Marcosende, ments also provides a satisfactory alternative for the reuse 36310 Vigo, Pontevedra, Spain of waste materials [6].

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One of the classic amendments used for recovering through combining plant tolerance traits with effective degraded or contaminated soils is compost. Using amend- amelioration of the root zone. The success of the phytosta- ments produced by composting to increase the organic bilisation of tailings landscapes depends on the success of content of degraded soils is a classic technique, which is engineered pedogenesis (or soil formation) in the tailings currently being perfected using higher-quality compost. amended with different organic and inorganic materials, These types of amendments can decrease metal bioavail- and the development of root zones (or soil subsystems) ability, shifting them from ‘‘plant-available’’ forms, which [16]. According to the literature, some authors such as Hat- are extractable with water or solutions of neutral salts such tab et al. [17] carry out experiments on degraded or con- as CaCl2, to fractions associated with organic matter, car- taminated soils in posts or using small quantities in the lab- bonates, or metal oxides [7]. Moreover, in this way these oratory, and study in top soils (0–25 cm). For this reason, materials are no longer considered as waste, and a value- in this study our aim was to scale up these experiments. added product is generated [8]. However, there are some In this study, we also determine the capacity of biochar inconveniences associated with the use of compost, one to fix metals and to improve the positive effects and reduce of which is the low recalcitrant value of the carbon that is the possible negative effects of compost, supplemented provided, or that it may contain some metals [9]. For this with Brassica juncea L. plants. B. juncea L. has been used reason, at present it is combined with biochar for reducing successfully for the phytoremediation of metals from pol- these potential problems [10]. luted mine soils [18–20]. According to some authors such Biochar is the solid product from pyrolysis of waste as Rahman et al. [21], Cu, Pb, and Zn are particularly biomass residues. Typically, most of the carbon in biochar important, as high quantities of these metals can decrease has an aromatic structure that is highly recalcitrant in the crop production due to the risk of biomagnification and environment, a high pH value, and cation exchange capac- bioaccumulation in the food chain, also effect on the plant ity, and is capable of enhancing productivity [11]. Being community and the consequent effect on soil physical deg- used as a soil amendment, biochar often has a liming effect radation, transport of pollution, and the consequent effect and a microporous structure, resulting in a high specific on the microbial and animal communities. In this experi- surface area and active functional groups on the surface, ment, we chose to study Cu because it is present at high which implies a high capacity for complex metals on their concentrations in the settling pond soil, and Ni, Pb, and surface. The surface sorption of metals on biochar has been Zn because they are present at considerable concentra- demonstrated on multiple occasions using scanning elec- tions in the compost used. Another objective was to find tron microscopy [10, 12]. If biochar is applied to soil, the out how B. juncea L. can strengthen and enhance the effect negative charge and, hence, the cation exchange capacity of treatments made with two different organic amendments (CEC) may increase, due to the induced higher pH, and, (compost and biochar). This study was carried out over an in turn, the electrostatic attraction between the metal cati- 11-month period in the settling pond of a depleted copper ons and soil particles will become stronger. Biochar can mine located in Touro (in Galicia, north-west Spain). We stabilise metals in contaminated soils, improve the quality put the settling pond soil into 50-cm cylinders, for repro- of the contaminated soil, and lead to a significant reduc- ducing as closely as possible the first few centimetres of the tion in the uptake of metals by crops [11]. Therefore, the settling pond in the field. The settling pond soil has been application of biochar can potentially provide a new solu- treated with various combinations of compost and biochar, tion for the remediation of the soils contaminated by metals and B. juncea L. has been planted. The effects of the treat- and can enhance soil productivity [11, 13, 14]. The reduced ments were studied at three different depths over a length bioavailability of metals and other modifications to the sub- of 45 cm. strate brought about by applying biochar may be beneficial to the establishment of plant cover on top of the waste, in order to achieve long-term phytostabilisation [5]. Materials and Methods Apart from using classic amendments such as compost, or more modern versions such as biochar, these are com- Soil Sampling bined with the use of phytoremediation plants. Pilon-Smits [15] interestingly defined phytoremediation as ‘the use The sampling zone is located in an old copper mine in of plants and their associated microbes for environmen- Touro, north-west Spain (8° 20′ 12.06″W 42° 52′ 46.18″N) tal cleanup’, which is an alternative, cost-effective, non- (Fig. 1). The climate in this zone is Atlantic (oceanic) with invasive technology for engineering-based remediation precipitation reaching 1886 mm per year (with an aver- methods. One type of phytoremediation is phytostabilisa- age of 157 mm per month) and a mean daily temperature tion. Phytostabilisation has been proposed as a sustainable of 12.6 °C. The average relative humidity is 77% (AEMET, option to stabilise and remediate base metal mine tailings 2015). One soil and three amendments were selected: the

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control, compost (C) supplied by the company Ecocelta Galicia S.L. (Ponteareas, Pontevedra, Spain), and biochar (B) provided by the company PROININSO S.A. The settling pond soil (S) comprised waste material resulting from the flotation of sulphides during copper processing. Due to the large amounts of soil necessary to develop the experiment, soil was collected with shovels and transported in polyethylene containers. The pool has been dry for several years and is considered to be a soil classified as Technosol according to the latest version of the FAO [22]. The sand (SS) was washed sea sand. The compost (C) consisted of horse and rabbit manure mixed with grass cuttings, fruit, and seaweed. The biochar (B) used was made from Quercus ilex wood with a pyrolysis temperature of 400 °C for 8 h.

Greenhouse Experiment

The greenhouse experiment was carried out in cylinders to try to reflect the top 45 centimetres of the soil; the cylinders were made of PVC, measuring 50 cm high with a diameter of 10 cm. A porous mesh was inserted into the cylinders, and the settling pond soil was poured inside. The mesh used for the settling pond soil was not in contact with the PVC for a long period of time (Fig. 2). The cylinders were filled with settling pond soil (S, negative control), settling pond soil and sand (SS, sand control), and settling pond soil with different treatments: Settling pond soil + compost (SC), Settling pond soil + compost + vegetated with B. juncea L. (SCP), Settling pond soil + compost + biochar Fig. 1 Location of the sampled area in Touro mine (SCB), and Settling pond soil + compost + biochar + vegetated with B. juncea L. (SCBP). The total weight of each soil chosen belongs to the settling pond (S) at the Touro cylinder was 3.5 kg. The amendment ratios used are shown mine, and the three amendments were as follows: sand in Table 1. The experiment was carried out over 11 months (SS) provided by the company Leboriz S.L.U., as a neutral at a controlled temperature and humidity (at a temperature

Fig. 2 Cylinder design and the different heights

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Table 1 Proportions used to prepare the controls and different treat- The plants were harvested in the same state of maturity, ments for comparison in the same state of development at three Soil (%) Sand (%) Compost (%) Biochar (%) different times (Time 1—3 months, Time 2—7 months, and Time 3—11 months). Growth was allowed under S 100 greenhouse-controlled conditions, with a photoperiod of SS 85 15 11:13 light/dark, a temperature of 22 ± 2 °C, and 65 ± 5% SC 85 15 relative air humidity. At the end of each time period, the SCP 85 15 roots and shoots were divided and carefully washed with SCB 85 11 4 deionised water. Fresh biomass was weighed immedi- SCBP 85 11 4 ately, and dry mass was assessed after oven drying for 48 h at 80 °C and cooling at room temperature. The plant tissues, divided into roots and shoots, were air dried and of 22 ± 2 °C and 65 ± 5% relative air humidity). A total of ground. The total contents of Cu, Ni, Pb, and Zn in B. 90 cylinders (15 cylinders per treatment) were prepared juncea L. were extracted by acid digestion using a mix- and randomly distributed (S, SS, SC, SCP, SCB, SCBP) ture of H 2O2 and HNO 3 (1:6v/v) in a microwave oven (Fig. 2). Three cylinders of each type were withdrawn at 3 (Milestone ETHOS 1). different times: Time 1—3 months, Time 2—7 months, and In this experiment, different factors were calculated in Time 3—11 months. The meshes were removed from the order to determine the phytoremediation capacity of B. cylinders and processed for analysis at 3 different depths: juncea L. the first from 0 to 15 cm, the second from 15 to 30 cm, and The transfer coefficient (TC) of the plants represents the third from 30 to 45 cm (Fig. 2). The cylinders were their efficiency to take up metals from the soil. The plant watered with drop water to field capacity throughout the is considered to be an accumulator–hyperaccumulator experiment. biosystem when TC >1 [27]. TC was calculated using the following equation: TC = Cp∕Cso, Soil Analysis where TC represents the transfer coefficient of the shoots, The settling pond soil samples collected from the cylin- Cp is the metal concentration in the shoots (mg kg− 1), and ders were air dried, passed through a 2-mm sieve, and Cso is the metal content of the soil (mg kg− 1) [28, 29]. homogenised prior to the analysis. Soil pH was determined A high value of the translocation factor (TF) indicates using a pH electrode in water to soil extracts with a ratio a relatively high shoot metal concentration compared to of 1:2.5 [23]. Total soil carbon (TC) and total nitrogen its root concentration. A plant species translocates metals (TN) were determined in a LECO CN-2000 module using effectively from the roots to shoots when TF >1 [30]. TF solid samples. Exchangeable cations were extracted with is expressed by the following equation: 0.1 M BaCl2 [24] and their concentrations determined by TF = Cs∕Cr, ICP-OES (Optima 4300 DV; Perkin-Elmer). Phytoavail- able contents of Cu, Ni, Pb, and Zn were extracted with where Cs and Cr are the metal concentrations (mg kg− 1) in 0.01 M CaCl2 in soil solution [20, 25]. Pseudo-total metal shoots and roots, respectively. contents were extracted with aqua regia by acid digestion in a microwave oven (Milestone ETHOS 1, Italy). Metal concentrations were determined by ICP-OES (Optima 4300 Statistical Analysis DV; Perkin-Elmer). Pseudo-total concentrations were compared with the background levels established for Galician All of the analytical determinations were performed in soils [26]. triplicate. The data obtained were statistically treated using the SPSS program version 19.0 for Windows. Analysis of variance (ANOVA) and test of homogeneity Plant Growth and Determination of Metals in Plant of variance were carried out. In case of homogeneity, a Tissues post hoc least significant difference (LSD) test was carried out. If there was no homogeneity, Dunnett’s T3 test B. juncea L. were pre-germinated in seedbeds until the was performed. Student T test was used to compare the growth of two fully expanded leaves, and were then trans- results of TC and TF between SCP and SCBP and the ferred the cylinders with one plant for each (SCP, SCBP).

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CaCl2-extractable contents of Cu, Pb, Ni, and Zn between have detectable CaCl 2-extractable concentrations of Cu SCP and SCBP. and Pb (Table 2).

Evolution of the Pseudo‑total Contents of Cu, Pb, Results Ni, and Zn at the Three Different Depths and Over the 11‑Month Period General Characteristics of Settling Pond Soil (S), Sand (SS), Compost (C), and Biochar (B) The settling pond soil (S) had the highest Cu concentration at depths 0–15 cm and 15–30 cm (P < 0.05, Fig. 3a, b, c, The soil from the settling pond soil (S) and the neutral d). At depth 0–15 cm, the SC and SCP treatments had the control (sand, SS) had an acidic pH, while the compost highest Pb concentrations (Fig. 3a, b). The Ni concentra- and biochar (C and B) had higher pH values (P < 0.05) tions were higher in SCBP at Time 1, and in SCP at Time (Table 2). The biochar had the highest pH (Table 2). 3 (P < 0.05, Fig. 3a, b). At Time 1, the Zn concentrations Total carbon (TC) was significantly higher in the bio- were higher in SC, while at Time 3 these concentrations char compared to the soil from the settling pond and were significantly higher in SCP (Fig. 3a, b). At depth sand (P < 0.05) (Table 2). The compost had the highest 15–30 cm and at Time 1, SCBP had higher concentrations exchangeable cation capacity (CEC) and total nitrogen of Pb and Ni, while the highest concentrations of Zn were content (TN) (P < 0.05) (Table 2). TN was undetectable observed in SC (P < 0.05, Fig. 3c). At Time 3, SCBP once in the settling pond soil (S) (Table 2). The pseudo-total again had the highest Pb concentrations (Fig. 3d). Both concentration of Cu in the settling pond soil was higher SCP and SCB had higher Ni concentrations in comparison than that in the amendments used (C and B) (P < 0.05) to the other treatments and controls (P < 0.05). The high- (Table 2). The settling pond soil had the highest pseudo- est Zn concentrations were found in the neutral control total concentration of Cu. The compost had the highest (SS) and SCP (P < 0.05, Fig. 3d). At depth 30–45 cm and at pseudo-total concentration of Pb, Ni, and Zn, especially Time 1, SCBP had the highest concentrations of Cu. Both Zn (Table 2). S and C had pseudo-total Cu concentra- the neutral control and the settling pond soil treated with tions that are above the background levels established compost + biochar + B. juncea L. had the lowest concentra- for Galician soils (Table 2), while the pseudo-total Zn tions of Pb. The treatments that combined compost and bio- concentrations in C exceed the background levels estab- char had the highest Ni concentrations (P < 0.05), while the lished for Galician soils (Table 2). The CaCl2-extractable highest Zn concentrations were observed in SCP (P < 0.05, concentrations of Cu, Pb, and Ni in S were significantly Fig. 3e). At Time 3, SC had the highest concentrations of higher than those in the C and B (P < 0.05) (Table 2). The Cu (Fig. 3f). As regards the Pb concentrations, the highest CaCl2-extractable concentration of Zn in C was higher values were found in SCP, which also had the highest Ni than that in S, SS, and B (Table 2). The biochar did not concentrations, as well as SCB (P < 0.05, Fig. 3f). Finally,

Table 2 Selected S SS C B BL characteristics and metal concentrations of settling pond pH 2.73 ± 0.08d 3.78 ± 0.14c 6.25 ± 0.04b 9.83 ± 0.16a soil (S), sand (SS), compost (C), Total Carbon (g/kg) 1.93c 2.76c 276 ± 2.66b 675 ± 5.50a and biochar (B) Total Nitrogen (mg/kg) u.d 0.10c 21.3 ± 1.02a 5.34 ± 0.10b − 1 CEC (cmol(+)kg ) 6.10c 0.13 ± 0.05d 53.5 ± 1.07a 15.6 ± 0.63b Cu Pseudo-total (mg/kg) 437 ± 1.74a 46.6 ± 0.96c 193 ± 1.14b 27.2 ± 1.29d 50 Pb 17.7 ± 0.41b 10.1 ± 0.27c 26.6 ± 0.96a u.d. 80 Ni 10.8 ± 0.97c 8.41 ± 0.52c 49.7 ± 1.71a 25.3 ± 0.79b 75 Zn 64.7 ± 1.28b 18.7 ± 0.85c 403 ± 3.33a 62.4 ± 1.70b 200 Cu 163 ± 3.84a 3.13 ± 0.36b 0.95 ± 0.04b u.d.

Pb CaCl2 (mg/kg) 9.56 ± 1.37a 0.14 ± 0.03b 0.14 ± 0.01b u.d. Ni 0.55 ± 0.03a 0.05 ± 0.02c 0.24 ± 0.03b 0.33 ± 0.02b Zn 2.89 ± 0.01b 0.32 ± 0.04d 7.98 ± 0.05a 1.25 ± 0.05c

For each row, different letters in different samples mean significant differences (n = 3, ANOVA; P < 0.05). u.d. undetectable level. Typical deviation is represented by ± BL (background levels) established for Gali- cian soils [26]

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Fig. 3 Evolution of the pseudo-total concentrations of Cu, Pb, Ni, Settling pond soil (S), settling pond soil + sand (SS), settling pond and Zn at three depths and over the 11-month period. For each row, soil + compost (SC), settling pond soil + compost + Brassica juncea different letters in different samples mean significant differences L. (SCP), settling pond soil + compost + biochar (SCB), settling pond (n = 3, ANOVA; P < 0.05). Error bars represent standard deviation. soil compost + biochar + Brassica juncea L. (SCBP)

the highest Zn concentrations were observed in the neutral had a higher CaCl2-extractable Pb concentration than in control (SS) (Fig. 3f). the treated settling pond soil, and SCP had the highest values of CaCl2-extractable concentrations of Cu, Ni, and Zn Evolution of the CaCl2‑Extractable (Phytoavailable) (Table 4). At Time 2, S had the highest CaCl 2-extractable Contents of Cu, Pb, Ni, and Zn at the Three Different Cu concentrations. The highest Pb concentrations were Depths and over the 11‑Month Period observed in S and SCP (P < 0.05). In turn, both S and SCBP had the highest Ni concentrations, and SCP had the At depth 0–15 cm, the settling pond soil (S) had the higher highest Zn concentrations (P < 0.05) (Table 4). At Time CaCl2-extractable Cu, Pb, and Ni concentrations at the 3, the controls (S, SS) had the highest concentrations of three different times than in the treated settling pond soil; CaCl2-extractable Cu, while the negative control (S) and SC had the highest CaCl 2-extractable concentration of Zn SC had the highest concentrations of CaCl 2-extractable at Time 1, but at Times 2 and 3 the SCP treatment had Pb (Table 4). SCBP had the highest CaCl2-extractable the highest CaCl 2-extractable concentration of Zn, a con- concentrations of both Ni and Zn (P < 0.05, Table 4). At tent significantly equal to S at Time 1 (P < 0.05, Table 3). the depth of 30–45 cm and at Time 1, S had the highest At depth 15–30 cm and at Time 1, the settling pond soil CaCl2-extractable Pb concentration (Table 5). SCP had the

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Table 3 CaCl2-extractable S SS SC SCP SCB SCBP concentrations of Cu, Pb, Ni, and Zn after different treatments Time 1 at depth 0–15 cm and at three Cu 50.6 ± 4.09a 3.37 ± 0.82b 0.90 ± 0.03c 0.75 ± 0.0.01c 0.87 ± 0.05c 0.74 ± 0.02c different times Pb 0.41 ± 0.02a 0.15 ± 0.02b 0.14 ± 0.01b 0.10c 0.12b 0.13 ± 0.01b Ni 2.06 ± 0.66a 0.070000 0.11c 0.08d 0.09 ± 0.01d 1.27 ± 0.37b Zn 3.09 ± 0.07e 0.42 ± 0.01f 7.51 ± 0.02a 6.3 ± 0.10b 5.71 ± 0.03c 4.72 ± 0.42d Time 2 Cu 54.5 ± 3.94a 5.31 ± 045b 0.61 ± 0.10c 0.60 ± 0.01c 0.17d 0.58 ± 0.89c Pb 0.44 ± 0.05a 0.15 ± 0.01b 0.15b 0.10 ± 0.02c 0.06d 0.11 ± 0.03c Ni 2.53 ± 0.65a 0.02 ± 0.01d 0.09c 0.11 ± 0.03c 0.08 ± 0.02c 0.67 ± 0.12b Zn 2.63 ± 0.07d 0.59 ± 0.39e 6.13 ± 0.32b 9.05 ± 2.01a 4.56 ± 1.00c 6.05 ± 0.89b Time 3 Cu 54.1 ± 3.22a 4.76 ± 0.89b 0.34 ± 0.02c 0.51 ± 0.03c 0.30c 0.46 ± 0.01c Pb 0.44 ± 0.13a 0.19 ± 0.03b 0.14c 0.11 ± 0.03d 0.14 ± 0.01c 0.15c Ni 3.32 ± 1.01a 0.02d 0.04 ± 0.01c 0.81 ± 0.05b 0.01d 0.04 ± 0.02c Zn 2.34e 0.31 ± 0.04f 12.0 ± 0.99b 16.1 ± 2.87a 7.86 ± 0.08c 6.92 ± 0.47d

− 1 CaCl2-extractable Cu, Pb, Ni, and Zn concentrations (mg kg ). For each row, different letters in different samples mean significant differences (n = 3, ANOVA; P < 0.05). Typical deviation is represented by ±. Settling pond soil (S), settling pond soil + sand (SS), settling pond soil + compost (SC), settling pond soil + compost + Brassica juncea L. (SCP), settling pond soil + compost + biochar (SCB), settling pond soil compost + biochar + Brassica juncea L. (SCBP)

Table 4 CaCl2-extractable S SS SC SCP SCB SCBP concentrations of Cu, Pb, Ni, and Zn after different treatments Time 1 at depth 15–30 cm and three Cu 50.9 ± 0.07b 50.2 ± 0.05b 42.0 ± 0.02c 54.6 ± 1.01a 41.0 ± 0.06d 40.8 ± 0.01e different times Pb 0.40a 0.38 ± 0.01b 0.36c 0.36c 0.31 ± 0.02d 0.32 ± 0.03d Ni 2.06 ± 0.01d 2.09 ± 0.04d 2.06d 3.60 ± 0.05a 2.82 ± 0.07c 2.95 ± 0.03b Zn 2.37 ± 0.05c 2.33 ± 0.02c 2.07 ± 0.10d 3.76 ± 0.03a 3.22 ± 0.01b 3.20 ± 0.04b Time 2 Cu 55.4 ± 0.06a 53.3 ± 0.85b 45.1 ± 0.33d 43.8 ± 0.02e 47.7 ± 0.02c 46.3c Pb 0.48 ± 0.04a 0.35c 0.37b 0.45 ± 0.04a 0.37 ± 0.01b 0.31 ± 0.02d Ni 3.44 ± 0.13a 2.38 ± 0.03c 2.39 ± 0.01c 2.97 ± 0.02b 2.94 ± 0.05b 3.44 ± 0.13a Zn 2.59 ± 0.01c 2.66 ± 0.02b 2.36 ± 0.01e 2.82 ± 0.11a 2.44 ± 0.03d 2.29 ± 0.04f Time 3 Cu 55.9 ± 0.07b 57.2 ± 0.02a 48.0 ± 0.05e 49.9 ± 0.04d 51.0c 48.1 ± 0.06e Pb 0.43 ± 0.01a 0.37d 0.43 ± 0.01a 0.41b 0.40 ± 0.01bc 0.39c Ni 3.31 ± 0.04b 2.48 ± 0.11e 3.12c 2.73 ± 0.51d 3.30 ± 0.09b 3.68 ± 0.07a Zn 2.58c 2.64 ± 0.04c 2.68c 2.74 ± 0.02b 2.00 ± d 2.95 ± 0.06a

highest CaCl2-extractable concentrations of Cu, Ni, and Zn. of Ni and Zn at Time 1 (P < 0.05, Table 5). The highest At Time 2, once again the settling pond soil had the high- CaCl2-extractable concentrations of Cu were found in SC est CaCl2-extractable concentrations of Pb, in the same way (Table 5). as SCP had the highest CaCl2-extractable concentrations

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Table 5 CaCl2-extractable S SS SC SCP SCB SCBP concentrations of Cu, Pb, Ni, and Zn after different treatments Time 1 at depth 30–45 cm and three Cu 52.0 ± 0.81c 53.6 ± 0.11c 54.2 ± 0.01b 57.7 ± 1.22a 53.9 ± 0.31bc 54.3 ± 0.05b different times Pb 0.55 ± 0.40a 0.43c 0.35 ± 0.01d 0.48 ± 0.02b 0.36 ± 0.01d 0.33e Ni 2.16 ± 0.09 f 2.26 ± 0.02e 2.78 ± 0.03d 4.08 ± 0.60a 3.55 ± 0.05c 3.75 ± 0.01b Zn 3.45 ± 0.03b 2.55 ± 0.20d 3.11 ± 0.01c 3.54 ± 0.04a 3.09 ± 0.02c 2.10e Time 2 Cu 53.1 ± 0.01c 52.7 ± 0.03c 56.9 ± 0.93a 49.1d 54.3 ± 0.05b 52.9 ± 0.02c Pb 0.55 ± 0.03a 0.42 ± 0.01 cd 0.38 ± 0.03d 0.45 ± 0.01b 0.43c 0.33e Ni 2.57 ± 0.06e 2.82 ± 0.01d 2.90 ± 0.03d 3.76 ± 0.06a 3.37 ± 0.11c 3.59 ± 0.05b Zn 3.43 ± 0.02b 3.00 ± 0.12c 3.32 ± 0.07bc 3.74 ± 0.04a 2.20 ± 0.07d 2.00 ± 0.18d Time 3 Cu 58.3 ± 0.57b 61.8 ± 0.99a 57.8 ± 1.23b 48.4 ± 1.08d 53.1 ± 0.01c 47.4 ± 0.04d Pb 0.47b 0.49a 0.40 ± 0.02d 0.44 ± 0.01c 0.49 ± 0.01a 0.31 ± 0.03e Ni 3.48 ± 0.01b 3.14 ± 0.02d 3.51 ± 0.01a 3.20 ± 0.04c 3.06 ± 0.02e 3.03 ± 0.05e Zn 3.73 ± 0.13a 3.27 ± 0.09b 3.02c 3.02 ± 0.03c 2.56 ± 0.04d 2.48 ± 0.01e

− 1 CaCl2-extractable Cu, Pb, Ni, and Zn concentrations (mg kg ). For each row, different letters in different samples mean significant differences (P b 0.05). Limit of detection = 5 × 10−5. Settling pond soil (S), settling pond soil + sand (SS), settling pond soil + compost (SC), settling pond soil + compost + Brassica juncea L. (SCP), settling pond soil + compost + biochar (SCB), settling pond soil compost + biochar + Brassica juncea L. (SCBP)

Harvestable Amounts of Cu, Pb, Ni, and Zn shoots of SCP, while SCBP had higher Zn concentrations and Determination of Metals in Plant Tissues: in the shoots than SCP. In the case of Ni in the shoots, there Translocation Factor (TF) and Transfer Coefficient were no significant differences. B. juncea L. harvested in (TC) SCP had the highest harvestable amounts of Cu, Pb, and Zn in their roots, although the harvestable amounts of Ni in the Harvestable Amounts of Cu, Pb, Ni, and Zn in Brassica roots were higher in SCBP (Fig. 4e, f, p < 0.05). juncea The highest harvestable amounts of Cu and Pb in the shoots were obtained at Time 2 in the plants harvested B. juncea L. did not grow in the settling pond soil (S) and from SCP, with the values of 0.07 mg/cylinder for Cu and in the sand control (SS), so it is not shown in Fig. 4a, b, c, 0.02 mg/cylinder for Pb. The highest harvestable amounts d, e, f. of Ni and Zn in the shoots were 0.04 mg/cylinder and In the first harvest (Time 1), the concentrations of Cu, 0.55 mg/cylinder respectively, which were obtained from Ni, and Zn in B. juncea L. shoots in the settling pond soil the plants harvested in SCBP at Time 1. As regards the har- treated with compost + biochar (SCBP) were significantly vestable amounts in the roots, SCP had the highest values higher than those in the settling pond soil that was only for Cu, Pb, and Zn, being 0.21 mg/cylinder, 0.04 mg/cyl- treated with compost (SCP); in the case of the harvestable inder, and 0.23 mg/cylinder, respectively, at Time 3. The amounts of Pb in the shoots, there were no significant dif- highest harvestable amount of Ni in the roots was 0.17 mg/ ferences between treatments. SCBP had the highest con- cylinder, which was obtained from the plants harvested in centrations of Ni and Zn in the roots of B. juncea L. that SCBP at Time 3. were harvested. The differences in the harvestable amounts of Cu in the roots were not significant, and those of Pb in Transfer Coefficient (TC) the roots were not detectable (Fig. 4a, b, p < 0.05). In the second harvest (Time 2), SCP had higher values of har- As occurred with the TF, the TC could not be calculated vestable amounts of Cu and Pb in the shoots than SCBP, for S and SS. The highest TC value for Cu was found in the and SCBP had higher harvestable amounts of Ni and Zn SCP treatment at Time 2, with a value of 0.22. The highest in the shoots than SCP. However, B. juncea L. harvested TC value for Pb was 0.55, in SCP at Time 1. SCBP had the in SCP had the highest harvestable amounts of Cu, Ni, and highest TC values for Ni and Zn, i.e., 0.65 and 0.82, respec- Zn in their roots, while the values of Pb in the roots were tively, at Time 1. At Time 1, the TC values for Cu and Pb not detectable (Fig. 4c, d). At Time 3, the highest harvest- were significantly higher in SCP than those in SCBP, but able amounts of Cu and Pb were once again found in the SCBP had the highest TC values for Ni and Zn (Fig. 5a). At

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Fig. 4 Harvestable amounts of Cu, Pb, Ni, and Zn and determina- tling pond soil + sand (SS), settling pond soil + compost (SC), set- tion of metals in plant tissues. For each row, differ letters in different tling pond soil + compost + Brassica juncea L. (SCP), settling pond samples mean significant differences (n = 3, Student’s t test: P < 0.05). soil + compost + biochar (SCB), settling pond soil compost + bio- Error bars represent standard deviation. Settling pond soil (S), set- char + Brassica juncea L. (SCBP)

Time 2, SCP had the highest TC values for Cu, Pb, and Zn. not grow on these soils. The highest TF values for Cu, In the case of TC for Ni, there were no significant differ- Pb, and Ni were obtained in SCBP at Time 1, with the ences between treatments (P < 0.05) (Fig. 5b). The TC val- values of 0.10, 0.19, and 0.11, respectively. The highest ues for Cu and Pb at Time 3 were significantly higher in the TF value for Zn was 0.55, also in SCBP, although this SCP treatment than those in the SCBP one. The TC values time at Time 3. The TF values at Time 1 for all the met- for Ni and Zn in SCBP were significantly higher than those als (Cu, Pb, Ni, Zn) were the highest in the settling pond in SCP (Fig. 5c). soil treated with compost and biochar (SCBP) (Fig. 6a). At Time 2, SCBP had a higher TF for Ni and Zn than Translocation Factor (TF) in SCP, and SCP had the highest Cu values (P < 0.05) (Fig. 6b). The TF values at Time 3 for Cu, Ni, and Zn The TF value could not be calculated for the S and SS were significantly higher in SCBP than those in SCP; for plants, because, as previously mentioned, the plants did Pb, the highest TF values were reported in SCP (Fig. 6c).

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Fig. 5 Transfer of Cu, Ni, Pb, and Zn to and within mustards accord- test: P < 0.05). Error bars represent standard deviation. Settling pond ing to transfer coefficient (TC) (shoot concentration/pseudo-total soil soil + compost + Brassica juncea L. (SCP), settling pond soil com- concentration) over the 11-month period. For each row, differ letters post + biochar + Brassica juncea L. (SCBP) in different samples mean significant differences (n = 3, Student’s t

Fig. 6 Translocation factor (TF) of Cu, Ni, Pb, and Zn to and within dent’s t test: P < 0.05). Error bars represent standard deviation. Set- mustards according Translocation factor (TF) (shoot concentration/ tling pond soil + compost + Brassica juncea L. (SCP), settling pond root concentration) over the 11-month period. For each row, different soil compost + biochar + Brassica juncea L. (SCBP) letters in different samples mean significant differences (n = 3, Stu-

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Discussion matter and increased pH produced by the treatments, as the pH and TC content in compost and biochar are very high. Changes in Pseudo‑Total Soil Cu, Pb, Ni, and Zn at the Three Different Depths and Over the 11‑Month Changes in CaCl2‑Extractable (Phytoavailable) Period Concentrations of Cu, Pb, Ni, and Zn at the Three Different Depths and Over the 11‑Month Period The high Cu content in the settling pond soil (S) is probably due to its source, as it is a soil from the settling pond of The effect of the different treatments was more visible at a copper mine, predominated by wastes resulting from the depth 0–15 cm, especially in the case of treatments SCB flotation of sulphides during copper processing. The set- and SCBP, which generally had the lowest values for the tling pond soil treated with compost and biochar (SCB and phytoavailable concentrations of Cu, Pb, and Ni. This is SCBP) had a lower Cu content at the end of the experiment due to the positive effect of the biochar when combined at the three depths. This is due, especially at the depth of with compost, as the positive effects of organic amend- 0–15 cm, to the dilution effect caused by the amendments ments are enhanced when combined with biochar [36]. used to prepare the treatments, as their pseudo-total Cu In turn, although the phytoavailable concentrations of Zn contents were much lower than those in S. Even so, after increased after applying the treatments to the settling pond treatment, both the settling pond soil and the sand control soil, if we compare the percentage of phytoavailable con- and the settling pond soil had pseudo-total contents that centrations in relation to the pseudo-total, these decrease to exceeded the background levels for Galician soils [26]. In 6.40% for S and an average of 2.87% for the different treat- general terms, the pseudo-total contents of Pb, Ni, and Zn ments. The positive effect of the compost is due to the ele- increased in the settling pond soil once the different treatments used to make it, such as waste from agri-food indus- ments had been applied to it. This increase occurred at all tries (horse and rabbit manures, grass cuttings). Several depths and is due to the presence of metals in the materi- authors have demonstrated that these wastes have a high als used to prepare the treatments, especially the compost, organic matter content and high concentrations of basic cat- as horse manure and seaweed can contain considerable ions. A direct effect of this high basic cation content is high pseudo-total metal concentrations [31, 32], or, as already CEC and pH [33, 34, 37]. Another component of the com- indicated by Canet et al. [33]’, rabbit manure may have as post used that influences the reduction of the phytoavail- much as 263 mg/kg of Zn. This increase in Zn concentra- able concentrations is seaweed. This is because biosorption tion was more distinct at the depth of 0–15 cm and even in algae has mainly been attributed to the cell wall, com- exceeded the background levels for Galician soils [26], posed of a fibrillar skeleton and an amorphous embedding although at the depths of 15–30 cm and 30–45 cm the matrix. Both electrostatic attraction and the complexation pseudo-total concentrations of Zn did not exceed the back- of metals in the biomaterial can play a role [38]. As pre- ground levels for Galician soils. viously mentioned, the positive characteristics of the com- In turn, although the pseudo-total concentrations of Pb post were improved by the biochar, as the biochar has a and Ni also increased, they did not exceed the background negative charge, and so the cation exchange capacity (CEC) levels for Galician soils at any of the depths. It should be may increase due to the induced higher pH and, in turn, the noted that despite the increase in the pseudo-total content electrostatic attraction. Biochar also has a large surface area of Cu, Pb, Ni, and Zn in the S soil due to the treatments, the and a high organic matter content [5, 10, 13,]. The SCB and percentage of phytoavailable concentrations in relation to SCBP treatments had lower phytoavailable concentrations the pseudo-total decreased. The average percentage of phy- of Zn than SC and SCP, revealing the ability of biochar to toavailable concentrations in relation to the pseudo-total for enhance the ability to reduce the phytoavailable concentra- S at depth 0–15 cm was 9.6% Cu, 1.51% Pb, 20.86% Ni, tions of the compost. and 6.40% Zn, while for the treated soils the averages were At depth 15–30 cm, the effect of the treatments did 0.30% Cu, 0.31% Pb, 1.17% Ni, and 2.87% Zn. This reduc- not follow the same pattern for all of the metals at all the tion in the percentage of phytoavailable concentrations in times. At Time 1, the settling pond soil to which the treat- relation to the pseudo-total of Cu, Pb, and Ni is due to the ments were applied generally had a higher phytoavailable ability of the compost and biochar to fix these metals [11, concentration of the metals that were studied, especially in 12, 34]. Some authors [35] found linear relations between the case of SCP. This increase is due to the metal inputs the activity or solubility of several metals and the soil prop- by the soil amendments [31–33]. However, over time, the erties such as pH, organic matter content, and pseudo-total treatment SCB (compost + biochar) began to take effect metal concentration, because the reduction in the percent- and reduce the phytoavailable concentrations of the metals age of phytoavailable concentrations in relation to the studied, both at Time 2 and Time 3, except for Ni. That this pseudo-total is possibly due to the contribution of organic effect was more detected over time is possibly due to the

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137 Waste Biomass Valor increased pH and organic matter content (the compost and values indicate that B. juncea L. could function as a phy- biochar had very high TC and pH values; Table 2), which tostabiliser for Cu, Pb, Ni, and Zn, as the ideal plant spe- play a crucial role in metal solubility, causing the metals to cies for phytostabilisation are the so-called metal excluders, be in a phytoavailable form in the soil [39–41]. which show a low root-to-shoot transfer coefficient [42]. At depth 30–45 cm, the positive effect of the compost As regards the low TF values, these indicate that instead and biochar combination became noticeable as time passed, of translocating these metals from the roots to the shoot, especially in the vegetated treatment (SCBP), which, on it fixes them in the roots [2]. This phytostabilising ability reaching the last time, had significantly lower concentra- of B. juncea L. is important, as phytostabilisation is the tions of trace elements in an assimilable form in compari- most appropriate method for relatively immobile materi- son with the untreated settling pond. This reduction of the als and large surface areas, and is currently acceptable for phytoavailable concentrations may be due to the increased remediation in mining sites [43]. The result is that exposure pH, TC, and CEC caused by the treatments, as the amend- to these metals by livestock, wildlife, and human beings is ments used had a high pH, CEC, and a high TC concen- reduced [44]. tration (Table 2). These increases in the TC, pH, and CEC have a positive effect on the reduction of the phytoavailable concentrations [39–41]. Also, root uptake can affect the reduction of this concentration. Conclusions

The applied treatments were able to reduce the phytoa- Harvestable Amounts of Cu, Pb, Ni, and Zn and Metal vailable concentrations of Cu, Pb, and Ni at the depth of Concentrations in Plant Tissues: Translocation Factor 0–15 cm throughout the experimental time. However, the (TF) and Transfer Coefficient (TC) treatments failed to reduce the phytoavailable concentra- As regards the harvestable amounts of Cu, Pb, and Ni in tions of Zn at this depth. The trend at depth 15–30 was the shoots of B. juncea L., a trend was observed during that the treatments applied worked better at Time 2 than at the experiment whereby B. juncea L. grown on the treat- Times 1 and 3. In these treatments at Time 2, SC, SCB, ments without biochar (SCP) was found to have higher and SCBP also reduced the phytoavailable concentrations concentrations of these metals (P < 0.05). This is possibly of Zn. At depth 30–45 cm at the end of the experimen- due to the fact that in SCP the metals would be in a more tal time (Time 3), the SCBP treatment showed the best available form: in fact, at depth 0–15 cm and at Time 3, results. The SCBP treatment had lower values than the out of the four metals studied, three of them have higher settling pond soil at the last depth at Time 3. Only at the CaCl -extractable concentrations in SCP than in SCBP last depth (0–45 cm) and at Time 3, a better behaviour of 2 B. jun- (P < 0.05). The lower CaCl -extractable concentration in the treatment elaborated with compost + biochar + 2 cea SCBP is due to the fact that the biochar enhances the sorp- L. (SCBP) was observed. This indicates that perhaps tion capacity of the metals [9]. The situation with Zn was it would be necessary to use a higher amount of biochar, or different, possibly because Zn is a metal with less affin- that the effect of biochar will be more evident in the long ity for organic amendments, as it tends to form external term because the biochar is a highly recalcitrant material. B. juncea sphere complexes with the organic matter, which are very The TC and TF results revealed that L. cultivated weak and unstable, and which as a result of variations in in these treatments did not present phytoextractive capac- B. juncea the pH may cause Zn to be released [35]. For the harvest- ity; however, L. presented good phytostabilising able amounts of Cu, Pb, Ni, and Zn, the same trend was capacity for Cu, Pb, Ni, and Zn. observed in the roots as in the shoots, especially at Times 2 and 3, where it was found that the roots of B. juncea L. grown in the treatments without biochar (SCP) had the References highest harvestable amounts of Cu, Pb, Ni, and Zn, except for Ni at Time 3. 1. Luna, L., Miralles, I., Andrenelli, M.C., Gispert, M., Pellegrini, The B. juncea L. plants that were cultivated in the set- S., Vignozzi, N., Solé-Benet, A.: Restoration techniques affect tling pond soil to which the different treatments were soil organic carbon, glomalin and aggregate stability in degraded soils of a semiarid Mediterranean region. Catena. 143, 256–264 applied (SCB, SCBP) would not have a phytoextraction (2016) capacity according to the TC and TF values for the studied 2. Nouri, M., Haddioui, A.: Human and animal health risk assess- metals, as these TC values were lower than 1 in all treatment of metal contamination in soil and plants from Ait Ammar ments [27]; also, according to [30], the values of TF <1 abandoned iron mine, Morocco. Environ. Monit. Assess. 188, 6 (2016) mean that the plant is not capable of translocating metals 3. Venkateswarlu, K., Nirola, R., Kuppusamy, S., Thavamani, effectively from the roots to shoots. In turn, these low TC P., Naidu, R., Megharaj, M.: Abandoned metalliferous mines:

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6. Anexos

6.1. Anexo I. Increasing the nutrient content in a mine soil through the application of technosol and biochar and grown with Brassica juncea L.

Anexo I

Increasing the nutrient content in a mine soil through the application of technosol

and biochar and grown with Brassica juncea L. (Waste and Biomass

Valorization, under review)

Rubén Forján*, Alfonso Rodríguez-Vila, Emma F. Covelo aDepartment of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, Lagoas,

Marcosende, 36310 Vigo, Pontevedra, Spain

*Corresponding author, Tel.: +34 986812630; fax: +34 986812556. E-mail: [email protected]

Abstract

Mining is an anthropogenic activity that causes a profound environmental impact in many parts of the world, including soil degradation through physical, chemical and biological transformations. Mine soils are nutritionally deprived habitats characterized by unfertile soil with extreme pH values, a low cation exchange capacity, low nutrient availability, and poor organic matter. Today, techniques such as the use of technosols and biochar are starting to be used with the aim of recovering these soils. In this experiment we will compare the nutrient supply, increased pH, total carbon, total nitrogen and cation exchange capacity of two treatments made of different amendments (technosol and biochar) on a mine soil. We will also determine the capacity of biochar to fix nutrients and enhance the positive effects of technosols in order to achieve the continuous growth of Brassica juncea L. The effects of the treatments were studied at three different depths over the 45-centimetre length of each cylinder. The study lasted a total of 11 months, using a settling pond from a depleted copper mine in Touro (Galicia, north-west Spain). The results of this experiment revealed that the treatments applied increased the pH, nutrient, total carbon, total nitrogen and cation exchange capacity values. In turn, in the majority of the factors studied, the treatment combining the technosol and biochar was the most effective, with the Brassica juncea L. grown on this treatment having the highest biomass values.

Key words: Technosol, Biochar, nutrients, mining, soil recovery, Brassica juncea L.

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1. Introduction

Mining is an anthropogenic activity that causes high environmental impact in many areas of the world, including soil modification and degradation through physical, chemical and biological transformations. The mining extraction process completely removes completely flora, fauna, and soils from the previous system. Following resource exhaustion, post-mine areas are typically characterized by low soil organic matter content, low fertility, and poor physio- chemical and biological properties, permanent changes to the topography and geological structures, and disruption to the subsurface hydrologic regime. The reclamation process involves replacing the overburden, grading it to the original contour, and spreading the topsoil to a depth of about 30 cm [1-3]. Mining activities generate large amounts of waste materials and tailings that are deposited on the surface in the form of mine spoil dumps. These are nutritionally deprived habitats characterized by unfertile soil with alterations of pH values, a low cation exchange capacity, low nutrient availability, and poor organic matter [4].

Plant establishment and soil recovery in degraded habitats may be slow to recover by natural successional processes without human intervention [1]. The importance of a plant cover is related to the improvement of the physical (e.g. structure), chemical (e.g. increase of organic matter and nutrients, immobilization of contaminants, decreased leaching) and biological (e.g. increase of microbial activity and diversity) characteristics of soil or wastes [5]. For this reason, organic wastes such as sewage sludge and refuse or manure compost can be used as soil amendments, and to a certain extent as a slow release nutrient source. The organic matter and nutrient content of some common organic materials can be used to reduce the availability of metals, in addition to remediating the physical and chemical properties of the spoils, and the provision of plant nutrients. The incorporation of organic amendments into contaminated mine soils has been proposed as a feasible, inexpensive and environmentally sound disposal practice

[6,7]. Recently, a number of studies have focused on technosols, some of them dealing with the restoration of degraded areas [2]. Technosols combine soils whose properties and pedogenesis are dominated by their technical origin. They contain a significant amount of artefacts

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(something in the soil recognizably made or strongly altered by humans or extracted from greater depths) or are sealed by technic hard material (hard material created by humans, having properties unlike natural rock) or contain a geomembrane. They include soils from wastes

(landfills, sludge, cinders, mine spoils and ashes), pavements with their underlying unconsolidated materials, soils with geomembranes and constructed soils. Technosols are often referred to as urban or mine soils [8]. Furthermore, technosols could be elaborated from wastes and employed in the subsequent regeneration of degraded or polluted soils. Thereby, these materials are no longer considered as waste and a value-added product is generated [9]. The use of technosols increases the concentration of nutrients, the pH, cation exchange capacity, and even duplicates the organic matter content [10,11].

Today, researchers are beginning to study the use of biochar in the recovery of contaminated soils, as when biochar is mixed with amendments such as technosols, better results are obtained [12, 13, 14]. Also, using traditional amendments combined with biochar makes it possible to reduce costs. To create biochar, organic materials (i.e. feedstocks) are heated to temperatures between 300 ◦C and 800 ◦C in a low oxygen environment [15].

According to [1] the feedstocks may include agricultural wastes, forestry wastes, wood pellets, or manures. The high temperatures used in pyrolysis induce molecule polymerization within feedstocks to produce aromatic and aliphatic compounds. This creates a stable product demonstrated to be a potential sink for atmospheric CO2 and beneficial soil amendment. Recent research [16,17] has found that biochar is of great importance in increasing soil carbon storage, improving soil fertility, as well as maintaining the balance of soil ecosystems, and could act as a kind of soil fertilizer or amendment to increase crop yield and plant growth by supplying and retaining nutrients. The increase in soil fertility has been attributed to the high surface area of feedstock biochar (200–300 m2 /g) and high cation exchange capacity (CEC) (27.7–222.4 mmolc/kg), which increases the water retaining capacity, nutrient sorption, and enhances plant growth by supplying and retaining nutrients. As biochar is highly recalcitrant, the effects of its application may be prolonged over a long period of time [18].

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Establishment of the vegetation, site colonization with ambient plant species and recovery of the ecosystem is considered an indication of remediation success. A reclaimed, soil is necessary for it to be able to sustain a stable vegetation cover, as this is very effective in reducing surface erosion because the roots bind the substrate. Also, vegetation can return a large proportion of percolating water to the atmosphere through transpiration, thus reducing the concentrations of soluble metals entering water courses [19]. Vegetation cover also goes a long way towards reducing the visual scars in the landscape caused by large-scale mining operations.

Successful revegetation may allow for recreational use of the land, and even agriculture or forestry if conditions are favourable [19, 20]. It is very difficult to achieve a stable vegetation layer in these types of soils, as apart from containing metals, they usually have unfavourable properties such as a lack of nutrients, virtually no organic matter, and an extreme pH [21]. In order to achieve a stable vegetation cover on a degraded soil, such as a mine, it is necessary to remedy these deficiencies. Site preparation practices have the potential to modify the physical and chemical properties of the soil, thereby influencing nutrient availability for the establishing plants. For this reason, the early stage of vegetation establishment is particularly crucial and requires good site preparation practices to maximise the chance of success of the revegetation process [22].

The novelty of this work is to know the effect of the application of amendments made from residues to a degraded soil along 45 cm of depth. To know these effects we proposed the following objectives. The main objective of this study is compared the nutrient supply, increased pH, total carbon, total nitrogen, and cation exchange capacity of two treatments made using different amendments (technosol, biochar) on a mine soil. One treatment only contained technosol, while the second treatment contained technosol and biochar. Another objective of this study was to determine the capacity of biochar to retain nutrients and enhance the positive effects of technosols in order to be able to establish a permanent crop of Brassica juncea L.

Brassica juncea L. has been used successfully in polluted mine soils [23-25]. With this aim, in this study we used soil from the settling pond of a copper mine into 50-cm cylinders in order to

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Anexo I mimic as closely as possible the first few centimetres of the settling pond in the field. The settling pond soil was treated with different combinations of technosol and biochar, and

Brassica juncea L. was planted. This study lasted a total of 11 months. The settling pond belongs to the depleted soil copper mine located in Touro (Galicia, north-west Spain).

2. Material and Methods

2.1 Soil sampling and amendments

The sample zone is located in an old copper mine in Touro, north western Spain (8º 20'

12.06'' W 42º 52' 46.18'' N) (Figure 1). The climate in this zone is Atlantic (oceanic) with precipitation reaching 1886 mm per year (with an average of 157 mm per month) and a mean daily temperature of 12.6ºC. The average of relative humidity is 77% (AEMET, 2015). In order to carry out the study, one soil and three amendments were selected. The soil that was chosen belongs to the settling pond (S) at the Touro mine, S was comprised of waste material resulting from the flotation of sulphides during copper processing. The pool has been dry since 1988, and is considered to be soil according to the latest version of the FAO [8].

The three amendments were:

- Sand (SS) consisted in washed sea sand provided by the company Leboriz S.L.U.

(control).

- Technosol (T) provided by the company Tratamientos Ecológicos del Noroeste

(T.E.N.). The technosol (T) consisted of a mixture of 60% purification plant wastes,

10% aluminium company wastes (from Padrón, La Coruña, Spain) 5% ash (from Ence,

a cellulose company in Pontevedra, Spain), 10% wastes from the agri-food industry

(canning companies and Ecogal), and 5% purification plant sand (sand fraction). The

percentages do not add up to 100%, due to the privacy policy of the companies. The

company provided a few indicative percentages. For this reason the sum of the

percentages is not 100%. The technosol was elaborated with the aforementioned

residues.

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- The biochar (B) provided by the company PROININSO S.A., the biochar (B) used was

made from Quercus ilex wood with a pyrolysis temperature of 400 °C for 8 h

Fig. 1 Location of the sampled area in Touro mine.

2.2 Greenhouse experiment

The greenhouse experiment was carried out in cylinders to try to reflect the top 45 centimetres of soil; the cylinders are made of PVC with a depth of 50 cm and a diameter of 10cm. A porous mesh was introduced into the cylinders, and the settling pond soil into the inner, mesh was used for the settling pond soil was not in contact with the PVC for a long period of time (Figure 2).

The cylinders are filled with: Settling pond soil (S, negative control) Settling pond soil and sand

(SS, neutral control), and the treatments:

- Settling pond soil + technosol (ST)

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- Settling pond soil + technosol + vegetated with Brassica juncea L. (STP)

- Settling pond soil + technosol + biochar (STB)

- Settling pond soil+ technosol+biochar+ vegetated with Brassica juncea L. (STBP).

Fig. 2 Cylinder design and the different depths.

In the elaboration of the treatments STB and STBP, the technosol and the biochar were mixed, later the mixture was deposited in the surface of the soil.The amendment ratios used are detailed in Table 1. The total weight of each cylinder was 3.5 kg.

Table 1. Proportions used to make the controls and the different treatments Soil Sand Technosols Biochar

S 100%

SS 85% 15%

ST 85% 15%

STP 85% 15%

STB 85% 11% 4%

STBP 85% 11% 4%

The experiment was carried out over 11 months at a controlled temperature and humidity

(temperature of 22±2 °C, and 65±5 % relative air humidity). A total of 90 cylinders, 15 cylinders of each treatment were prepared and distributed randomly (S, SS, ST, STP, STB,

STBP). Three cylinders of each type were withdrawn at 3 different times: Time 1= 3 months,

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Time 2= 7 months, Time 3= 11 months. The meshes were removed from the cylinders and processed for analysis at 3 different depths: the first from 0-15 cm, the second from 15-30 cm, and the third from 30-45 cm (Figure 2). The cylinders were watered to field capacity throughout the experiment.

2.3 Soil, technosol and biochar analysis

The settling pond soil samples collected from the cylinders were air dried, passed through a 2 mm sieve and homogenized prior to analysis. Soil pH was determined using a pH electrode in 1:2.5 water to soil extracts [26]. Total soil carbon (TC) and total nitrogen (TN) were determined in a LECO CN-2000 module using solid samples. Exchangeable cations were extracted with 0.1 M BaCl2 [27] and their concentrations determined by ICP-OES (Optima 4300

DV; Perkin-Elmer). Pseudototal metal contents were extracted with aqua regia by acid digestion in a microwave oven (Milestone ETHOS 1, Italy). Metal concentrations were determined by

ICP-AES (Optima 4300 DV; Perkin-Elmer). Pseudototal concentrations were compared with the generic reference level (GRL) established for Galician soils [28].

A series of critical values were assigned to each of the chemical parameters, based on the model of the SFCC (Soil Fertility Capability Classification) proposed by [29] and adapted by [30-32]. These were used to evaluate the limiting factors for plant production.

2.4 Harvested biomass and height of Brassica juncea L.

The Brassica juncea L. were pre-germinated in seedbeds until they grew two fully expanded leaves, and were then transferred to the cylinders (STP, STBP). The plants were harvested in the same state of maturity, for comparison in the same state of development in the three times (Time 1= 3 months, Time 2= 7 months, Time 3= 11 months). Growth was allowed under greenhouse-controlled conditions, with a photoperiod of 11:13 light/dark, temperature of

22±2 °C and 65±5 % relative air humidity. At the end of each time period, the height of the plants was measured, and they were carefully washed with deionised water. Fresh biomass was weighed immediately, and dry mass was assessed after oven-drying for 48 h at 80 °C and

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2.5 Statistical analysis

All of the analytical determinations were performed in triplicate. The data obtained were statistically treated using version 19.0 of the programme SPSS for Windows. Analysis of variance (ANOVA) and a test of homogeneity of variance were carried out. In case of homogeneity, a post-hoc least significant difference (LSD) test was carried out. If there was no homogeneity, Dunnett’s T3 test was performed. Principal component analysis (PCA) was also carried out. Student-T test was using to compare the results of biomass and height between STP and STBP.

3. Results

3.1 General characteristics of the settling pond soil (S), sand (SS), technosol (T), and biochar (B).

The soil from the settling pond (S) and sand neutral control (SS) had an acidic pH, while the technosol and biochar (T and B) had higher pH values (P < 0.05) (Table 2). The biochar had the highest pH (Table 2). Total carbon (TC) was significantly higher in the biochar and technosol (T) compared to the soil from the settling pond and sand (P < 0.05) (Table 2). The technosol had the highest total nitrogen content (TN) (P < 0.05) (Table 2). TN was extremely low in the original soil and in the sand, and was actually undetectable in the soil (Table 2). S had the highest Fe concentration (Table 2). With regard to the concentration of the different nutrients studied, the technosol had the highest concentration of Ca, Mg, Mn and Na (Table 2).

K concentrations were significantly higher in the biochar compared to the S, SS and T (P <

0.05) (Table 2). On the other hand, in relation to the cation exchange, the Al3+ content in S was significantly higher than in the amendments (P < 0.05) (Table 2).

The amendments had higher concentrations of Ca2+, K+, Mg2+ and Na+ than S and SS.

The cation exchange capacity (CEC) of T and B was significantly higher than in the controls (S

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Anexo I and SS). The pseudototal concentration of Cu in the settling pond soil was higher than in the amendments used (T and B) (P < 0.05) (Table 2). The technosol had the highest pseudototal concentration of Pb and Zn, especially Zn. The biochar and technosol had a higher pseudototal concentration of Ni than S and SS (Table 2).

Table 2. Characteristics of the mine tailing (S), sand (SS), technosoils (T) and biochar (B).

S SS T B

pH 2.73±0.07d 3.83±0.55c 6.04±0.05b 9.90±0.02a

TC g.kg-1 1.93±0.15c 2.76±0.60c 256±2.51b 676±4.58a

TN mg.kg-1 u.d 0.10±0.01c 17.6±0.50a 5.34±0.22b

Ca 13.3±0.02c 9.65±0.05c 7785±15.9a 531±8.48b

Fe 323±8.97a 0.42±0.02b 0.05b u.d

. -1 K mg kg 6.40±0.89c u.d 2687±87.8b 3243±23.1a

Mg 216±2.10c 1.83d 1997±25.1a 548±11.6b

Mn 5.71±0.22b 0.18c 93.3±0.16a 0.12c

Na 27.4±0.22c 7.95±0.05c 2805±13.3a 65.4±1.29b

Al3+ 2.32±0.02a 0.02b u.d u.d

Ca2+ 0.07c 0.05c 38.9±0.01a 2.66±0.02b

-1 K+ cmol(+)kg 0.02c u.d 6.88±0.01b 8.30±0.05a

Mg2+ 1.78±0.01c 0.01d 16.3±0.05a 4.51±0.09b

Na2+ 0.14±0.02c 0.04d 14.0±0.15a 0.34±0.04b

-1 ECC cmol(+)kg 4.33±0.05c 0.12±0.01d 76.0±4.80a 15.8±17.8b

Cu 637±2.08a 46.4±1.14c 226±5.13b 27.1±1.24d

Pseudototal Pb 16.1±1.00b 10.4±0.56c 89.6±1.52a u.d (mg.kg-1)

Ni 16.4±1.10b 8.26±1.05c 26.3±0.57a 25.0±2.00a

Zn 65.4±2.51b 18.9±1.20c 340±5.50a 62.6±1.95b

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level. Typical deviation is represented by ±.

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3.2 Evolution of the pH at the three depths and over the 11-month period.

At depth 0-15cm, treatments STB and STBP (the settling pond soil treated with technosol and biochar) had the highest pH over time (Figure 3A). At depth 15-30cm, at 3 and 7 months, the treatments ST and STB had a significantly higher pH than the untreated settling pond soil (P <

0.05) (Figure 3B). At 11 months all treatments had a higher pH than the negative control (S)

(Figure 3B). At depth 30-45, the ST at 3 months and the STB at 7 months had a higher pH than the negative control (S); at 11 months, the treatments with technosols and biochar (STB, STBP) and the STP had a significantly higher pH than the untreated settling pond soil (P < 0.05) (Fig.

3C).

3.3 Evolution of Total Carbon (TC) at three depths and over the 11-month period.

At depth 0-15cm, treatments STB and STBP (the settling pond soil treated with technosol and biochar) had the highest TC concentration over time (Figure 4A). At depth 15-

30cm and 30-45cm, at 3 and 7 months, treatments STB and STBP had a significantly higher TC concentration than the untreated settling pond soil (P < 0.05) (Figure 4B, 4C). At 11 months, at

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Anexo I depth 15-30 and 30-45cm and 3, the settling pond soil treated with the different treatments had a higher TC concentration than the negative control (S) (Figure 4B, 4C).

3.4. Evolution of Total Nitrogen (TN) at three depths and over the 11-month period.

The TN was only detected at depth 0-15cm and only in the treated settling pond soil. At

3 and 11 months, the treated settling pond soil only with technosol (ST) had the highest TN concentration (Figure 5). At 7 months, treatments STB and STBP had a significantly higher TN concentration than treatments ST and STP (without biochar) (P < 0.05) (Figure 5).

Figure 5. Evolution of the total nitrogen (TN) at depth 0-15 cm and over the 11-month period. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represent standar deviation.

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3.5. Evolution of the cation exchange capacity (CEC), base saturation (%), and aluminium saturation (%) at three depths and over the 11-month period.

At depth 0-15cm, the treated settling pond soil (ST, STP, STB, STBP) had the highest cation exchange capacity (CEC) over time (P < 0.05) (Table 3). At 3 months, the STP treatment had the highest CEC, but at 7 and 11 months the STB and STBP treatments had the highest

CEC respectively (Table 3). The base saturation (V%) was higher in the treated soil than in the controls (S, SS). The settling pond (S) and the neutral control (SS) had a higher aluminium saturation (Al%) than the treated settling pond soil (ST, STP, STB, STBP) (Table 3). At depth

15-30cm, at 3 months, S and STBP had the highest CEC. At 7 months, the highest CEC was shown by S, ST and STB, although at 11 months, S had the highest CEC (P < 0.05) (Table 3).

The highest level of V% at the three times was provided by the treated settling pond soil (ST,

STP, STB, STBP) (Table 3). The settling pond soil (S) had the highest aluminium saturation

(Al%) over time (Table 3). At depth 30-45cm, the settling pond soil treated with technosol+biochar+ Brassica Juncea L. (STBP) had the highest CEC between 3 and 7 months.

At 11 months, the untreated settling pond soil had the highest CEC (P < 0.05) (Table 3). The treatment STB had the highest base saturation at 3 and 7 months, but at 11 months the highest

V% was corresponded to ST (Table 3). At 3 and 7 months, the settling pond soil (S) had the highest aluminium saturation (Al%) (Table 3). At 11 months, the treated settling pond soil (ST,

STP, STB, STBP) had the highest Al% (Table 3).

At depth 30-45cm, the settling pond soil treated with technosol+biochar+ Brassica

Juncea L. (STBP) had the highest CEC between 3 and 7 months. At 11 months, the untreated settling pond soil had the highest CEC (P < 0.05) (Table 3). The treatment STB had the highest base saturation at 3 and 7 months, but at 11 months the highest V% was corresponded to ST

(Table 3). At 3 and 7 months, the settling pond soil (S) had the highest aluminium saturation

(Al%) (Table 3). At 11 months, the treated settling pond soil (ST, STP, STB, STBP) had the

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Anexo I highest Al% (Table 3).

Table 3. Evolution of the cation exchange capacity (CEC), base saturation (V%), and aluminium saturation (Al%) at three depths and over the 11-month period. S SS ST STP STB STBP

CEC -1 (cmol(+)kg ) 2.58±0.16c 0.26±0.01c 21.5±2.31b 35.3±1.07a 23.6±0.87b 23.4±1.45b V% 39.4 43.7 99.9 99.9 99.9 99.9

3 months Al% 60.5 56.2 0 0 0 0

CEC -1

15 cm 15 (cmol(+)kg ) 5.06d 0.50±0.01d 25.3±0.40b 23.2±0.04c 26.0±0.17b 31.0±1.17a

- V% 36.1 52.9 99.9 99.9 99.9 99.9

7 months Al% 63.8 47.0 0 0 0 0

Depth 0 Depth CEC -1 (cmol(+)kg ) 5.86c 0.54±0.07c 30.0±1.79ab 25.2±0.26b 31.9±0.08a 29.1±0.96b

11 11 V% 41.8 44.2 99.9 99.9 99.9 99.9

months Al% 58.1 55.7 0.06 0 0 0

CEC -1 (cmol(+)kg ) 4.78±0.46a 0.84±0.03d 1.32±0.10c 2.25±0.07b 1.52±0.02c 2.85±0.77b V% 36.3 27.8 75.7 76.3 77.3 62.8

3 months

Al% 63.6 72.1 24.2 23.6 22.6 37.1

CEC -1 30 30 cm (cmol(+)kg ) 4.12±0.83a 1.21±0.08b 3.46±0.08a 1.30±0.06b 1.62±0.18b 4.43±0.06a

- V% 37.5 47.6 43.5 50.7 75.5 47.9

7 months Al% 62.2 51.6 56.4 49.2 24.4 52.0

Depth 15 Depth CEC -1 (cmol(+)kg ) 11.0±0.12a 5.00b 3.14±0.03c 2.49±0.02d 1.20±0.18e 1.19±0.18e

11 11 V% 38.3 39.0 41.0 52.1 73.0 71.3

months Al% 61.6 60.9 58.9 47.8 26.9 28.6

CEC -1 (cmol(+)kg ) 2.26±0.52b 0.69b 0.56±0.10b 0.78±0.05b 0.49±0.10b 5.38±0.33a 3 V% 41.1 26.8 60.1 54.7 66.3 34.6

months

Al% 58.8 73.11 39.8 45.2 33.6 65.3 CEC

45 45 cm -1

- (cmol(+)kg ) 4.60±0.09b 1.53±0.02c 4.06±0.18b 1.38±0.01c 1.65±0.01b 6.11±0.34a V% 32.8 59.5 42.9 52.0 58.7 35.4

7 months Al% 67.1 40.4 57.0 47.9 41.2 64.5

Depth 30 Depth

CEC -1 (cmol(+)kg ) 11.5±0.01a 7.29±1.71b 3.93±0.12c 3.19c 0.90±0.07d 0.93d

11 11 V% 40.2 33.9 39.9 37.4 35.6 38.4

months Al% 59.7 66.0 60.0 62.5 64.3 61.5 For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level. Typical deviation is represented by ±.

3.6. Evolution of nutrients in three depths and over the 11-month period.

At depth 0-15, the settling pond soil had the highest Fe concentration over time. The settling pond soil treated with the different treatments had higher Ca, K, Mg, Mn, Na concentrations than S over time, except at 11 months where Mn concentrations were lower in

STB and STBP than in S (P < 0.05) (Table 4.1).

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Table 4.1. Evolution of nutrients concentration (mg.kg-1)in three depths and along the 11 months

Depth 0-15cm S SS ST STP STB STBP

Ca 6.53±0.26b 4.61±0.81b 2837±310a 3847±330a 3282±100a 2771±161a

Fe 8.71±0.57a 1.49±0.61b 0.18±0.04c 0.49±0.17c 0.53±0.05b 0.06±0.05c

K 7.89±0.41c 6.46±0.23c 593±56.2b 1048±27.1a 643±42.1b 991±62.7a

3 months Mg 11.4±0.09c 2.07±0.29d 551±61.4b 996±1.19a 587±25.0b 581±35.9b

Mn 0.94±0.02d 0.14±0.01e 70.6±5.78a 95.9±7.75a 5.72±0.22b 4.11±0.19c

Na 21.7±0.51d 7.69±0.75e 247±21.9b 1043±129a 141±12.9c 458±37.5a

Ca 38.7±1.05c 16.5±2.20d 3690±64.6ab 2992±13.9b 3740±24.0a 3607±135b

Fe 696±42.9a 6.01b 0.92±0.29b 0.84±0.11b 0.07b 0.06±0.01b

K 2.12±0.51e 0.67±0.05e 481±4.56c 301±4.85d 615±1.27b 790±32.6a

7 months Mg 184±0.57c 7.49±0.20d 612±7.19b 645±2.89b 621±6.17b 904±39.9a

Mn 4.10±0.14d 0.51f 54.4±1.62b 59.5±0.35a 2.58±0.02e 5.53±0.05c

Na 17.8±1.24e 19.9±2.41e 130±1.57d 432±15.4b 138±0.99c 701±17.4a

Ca 58.4c 13.4±4.31d 4488±260.0a 3790±28.0b 4951±19.5a 4441±148.29ab

Fe 143±3.00a 4.88±1.52b 0.85±0.08c 0.36c 0.10±0.09c u.d

K u.d u.d 500±34.1a 191±1.23d 331±3.60b 225±5.42c

11 11 months Mg 253±1.63c 15.5±2.95d 698±45.4ab 628±12.4b 720±1.08a 704±22.8a

Mn 6.82±0.13c 0.72±0.08e 35.9±1.75b 42.3±0.16a 2.34±0.07d 2.32±0.15d

Na 11.9±0.48c 6.15±0.44d 115±7.60a 122±3.17a 84.7±0.50b 110±3.74a

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level. Typical deviation is represented by ±. At depth 30-15 cm, at 3 months the treatments with technosol and biochar had the highest nutrients concentration except for Na concentration. STP had the highest Na concentration (P < 0.05) (Table 4.2). At 7 months, STB and STBP had higher concentrations of

Ca and K than the controls (S, SS) (Table 3). The highest Mn concentrations were shown by the

ST, STP and STBP. The STBP and STB had the highest Na concentrations. The settling pond soil had the highest Fe concentration (Table 4.2). At 11 months, S had the highest Ca, Mg and

Mn concentrations. The ST and STP had the highest K and Na concentrations respectively. The neutral control (SS) had the highest Fe concentration (Table 4.2).

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Table 4.2. Evolution of nutrients concentration (mg.kg-1) in three depths and along the 11 months

Depth 15-30cm S SS ST STP STB STBP

Ca 8.18±0.80e 10.4±0.19e 57.0±5.12d 115±3.58b 98.1±3.61c 138±4.10a

Fe 6.09±0.32b 6.32±0.95b 3.73±0.36c 5.63±0.11b 7.32±0.16b 22.7±1.95a

K 14.7±0.29c 10.0±0.21d 58.0±6.35b 72.2±3.98b 95.9±1.21a 58.1±2.24b

3 months Mg 9.18±0.53d 9.82±0.77d 32.9±2.88c 56.8±2.10b 29.8±.024c 82.3±3.44a

Mn 0.96c 0.86±0.06c 2.04±0.15b 4.29±0.20a 1.63±0.13b 3.69±0.10a

Na 23.8±1.79d 10.8±0.67e 54.8±3.84b 93.3±4.53a 34.9±0.72c 50.3±1.42b

Ca 45.5±9.96b 50.2±9.54b 72.9±2.65b 48.9±0.46b 133±28.8a 98.6±1.25ab

Fe 487±110a 25.6±6.04c 108±0.54b 5.12±0.94c 3.79±0.35c 143±3.21b

K 0.54±0.42d 1.57±0.20d 7.45±0.24c 25.5±1.08b 88.8±3.06a 33.7±5.56b

7 months Mg 148±32.2a 21.6±0.49c 124±0.80b 17.4±0.64c 24.5±1.52c 152±1.38a

Mn 3.64±0.79bc 1.62±0.04b 6.74±.038a 1.79±0.02b 1.11±0.07b 5.57a

Na 16.7±2.33b 26.8ab 16.4±0.81b 36.7±1.39a 21.2±4.63b 53.9±8.62a

Ca 123±7.29a 34.1±0.66c 63.9±6.06c 128±13.5a 112±25.6b 115±26.3ab

Fe 47.7±0.80c 268±9.36a 62.4±0.01b 65.2±0.35b 8.26±0.54d 9.39±0.29d

K u.d u.d 1.22±0.06a u.d 1.56±1.06a u.d

11 11 months Mg 430±3.45a 207±1.72b 108±0.63c 69.7±0.89d 30.4±3.74e 25.3±2.98e

Mn 16.1±0.46a 4.68±0.06b 5.32±0.07b 4.99b 1.85±0.17c 2.52±0.33c

Na 11.5±0.58b 11.5±0.23b 10.7±0.08b 13.4±0.07a 8.93±1.07b 10.0±0.83b

Ca, Fe, Mg and Mn concentrations. The highest K concentrations were shown by STB and

STBP. The ST and STB had the highest Na concentrations (P < 0.05) (Table 4.3). At 7 months, the settling pond soil had higher Fe concentrations than the treated settling pond soil. The neutral control (SS) had the highest Ca concentrations. At 11 months, the settling pond soil (S) had higher Ca, Mg and Mn concentrations than the treated settling pond soil and the neutral control. SS had the highest Fe concentrations, and ST had higher Na concentrations than the controls and the other treated settling pond soils (P < 0.05) (Table 4.3).

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Table 4.3. Evolution of nutrients concentration (mg.kg-1)in three depths and along the 11 months

Depth 30-45cm S SS ST STP STB STBP

Ca 5.38±0.75c 11.5±0.06c 38.0±9.54b 66.4±0.40b 47.2±11.3b 118±0.42a

Fe 4.65±0.82b 6.12±1.28b 3.16±0.56b 6.06±0.02b 4.99±1.14b 300±3.18a

K 7.26±0.40c 9.98±1.95c 40.4±6.05ab 31.0±0.97b 72.5±21.0a 63.1±2.30a

3 months Mg 6.72±2.12c 12.7±0.01c 31.6±5.90b 36.1±0.47b 30.3±9.60b 231±9.13a

Mn 0.63±0.20c 1.42bc 1.97±0.33b 2.87±0.08b 1.09±0.24c 12.7±0.41a

Na 23.1±2.70b 9.05±0.67c 54.2±11.3a 43.9±1.76ab 55.3±19.9a 33.3±1.11b

Ca 54.1±10.1c 120±1.47a 74.5±1.25b 58.6±1.76bc 44.7±0.36c 75.9±0.47b

Fe 830±34.5a 20.6±0.95d 363±13.2b 8.12±0.57e 5.57±0.65e 276±15.1c

K 0.27±0.27d 2.01±1.70d 2.87±1.37d 18.9±0.68b 120±1.99a 14.1±1.07c

7 months Mg 144±4.57a 22.1±0.48b 150±6.26a 19.4±0.57c 17.5±0.16c 187±13.3a

Mn 3.42±0.06c 2.15±0.04d 4.07±0.14b 1.76±0.04e 0.68f 10.9±0.50a

Na 6.09±3.10c 20.6±6.19b 21.1±3.49ab 39.1±8.90a 55.2±0.74a 37.5±5.05a

Ca 150±0.26a 51.0±3.75b 32.7±1.59c 22.8±0.32d 18.7±2.05de 16.8±0.32e

Fe 218±7.40b 637±201a 105±3.15b 68.3±1.37b 14.1±0.57b 22.6±0.07b

K u.d u.d u.d u.d u.d u.d

11 11 months Mg 465±0.49a 260±40.1b 161±2.85c 122±0.58d 20.2±0.45e 26.6±.07e

Mn 12.7±0.12a 6.30±0.74b 3.99±0.10c 2.99d 1.53e 1.73±0.01e

Na 11.3±0.19b 12.0±0.77ab 12.6±0.25a 11.1±0.64b 9.10±0.95b 8.39±0.11c

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level. Typical deviation is represented by ±. The highest K concentrations were shown by the STB. The settling pond soil treated with the different treatments had higher Ca concentrations than the controls (Table 4.3). At 11 months, the settling pond soil (S) had higher Ca, Mg and Mn concentrations than the treated settling pond soil and the neutral control. SS had the highest Fe concentrations, and ST had higher Na concentrations than the controls and the other treated settling pond soils (P < 0.05)

(Table 4.3).

3.7 Limiting factors for plant production in depth 0-15cm.

All of the factors and values detailed above are shown in table 5. The high acidity of the settling

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Anexo I pond soil, over time, is a limiting factor for plant production (Factor c). The controls (S, SS) had deficient amounts of Ca that gave rise to the limiting Factor Ca (Table 5)

Table 5. Limiting factors for crop production linked to the content of bases in the complex change at depth 0-15 cm.

Factor c Factor Factor Factor Factor Factor Factor Ca e K Mg n N S 3.51 0.34 2.58 0.02 0.53 4.58 0 SS 4.08 0.02 0.26 0.02 0.01 17.9 0 ST 4.53 14.1 21.5 1.52 4.54 5.78 1.07 STP 5.70 19.2 35.3 2.68 8.20 14.7 1.86

3 months STB 7.31 16.4 23.6 1.65 4.84 3.03 1.02 STBP 7.17 13.8 23.4 2.54 4.79 9.7 1.19 S 3.02 0.19 5.06 0.01 1.52 1.95 0 SS 3.81 0.08 0.50 0 0.06 21.8 0 ST 6.55 18.4 25.3 1.23 5.04 2.59 0.99 STP 6.27 14.9 23.2 0.77 5.31 9.33 0.925

7 months STB 7.10 18.7 26.0 1.58 5.11 2.67 1.28 STBP 7.11 18.0 31.0 2.02 7.44 11.3 1.16 S 2.63 0.29 5.86 0 2.08 1.18 0

SS 3.33 0.07 0.54 0 0.12 7.43 0 ST 6.93 22.4 30.0 1.28 5.74 1.94 1.55 STP 7.03 18.9 25.2 0.49 5.17 2.46 1.98

11 months STB 7.14 24.7 31.9 0.85 5.92 1.35 1.64 STBP 7.48 22.2 29.1 0.58 5.79 1.93 1.36 -1 -1 -1 -1 Factor c pH<3.5; Factor Ca<1.5cmol(+)kg ; Factor e ECC <4cmol(+)kg ; Factor K <0.2 cmol(+) kg ; Factor Mg <0.4 cmol(+) kg ; Factor n Na> 15%; N Total content <0.1%

The CEC values are below the limit set by the Factor e at 3 months for the controls, and at 7-11 months only for the neutral control (SS) (Table 5). The K values were a limiting factor

(Factor K) in S and SS over time (Table 5). The neutral control (SS) had a deficiency in Mg2+ over time; this resulted in the presence of a limiting factor for plant production (Factor Mg)

(Table 5). Only SS at 3 and 7 months exceeds the limit set by the Factor n (Table 5). The settling pond soil and the neutral control are severely limited for plant production according to the Factor N over time (Table 5).

3.8 Harvested biomass of Brassica juncea L.

The Brassica juncea L. was not capable of growing in the settling pond soil (S) and in the neutral control (SS), and so it is not represented in figures 6A and 6B. The biomass of Brassica juncea L. harvested in treatment STPB was higher than in STP over time (Figure 6A; (P < 0.05).

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The Brassica juncea L. harvested in treatment STPB was the highest over time (Figure 6B; (P <

0.05).

Figure 6. Harvested biomass of Brassica Juncea L. over the 11-month period. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represents standar deviation

4. Discussion

4.1 Evolution of the pH at three depths and over the 11-month period.

The low pH of the settling pond soil (S) is due to its origin, as sulphide minerals in contact with water and air produce sulphuric acid [33], and also this soil has a predominance of wastes resulting from the flotation of sulphides during copper processing. At depth 0-15cm, after applying the treatments (ST, STP, STB, STBP) it was possible neutralize the acidity of the soil throughout the entire experiment. This was possibly due to the high pH of the amendments used in producing the treatments that were applied. The treatments that increased the pH the

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Anexo I most were those that combined technosol and biochar (STB, STBP). This is due, on the one hand, to the high pH of the elements used to create this technosol, something that has already been demonstrated by various authors, who have indicated that the types of waste used in this study have high concentrations of basic cations [34, 35]. This high pH of the technosol was increased by the biochar, which causes an increase in the pH of the soils where it is applied, as demonstrated by authors such as [36, 37]. At depths 15-30cm and 30-45cm, it was found that in the settling point soil (S) where the two different treatments were applied, the pH increased as time progressed. This is important, as it shows that these treatments do not only increase the pH in the top few centimetres, but also at depth. As occurred with depth 0-15cm, in depths15-30cm and 30-45cm a trend was observed in which treatments STB and STBP were more effective, further increasing the pH values. Soils with pH below 3.5 are strongly limited for plant production [38]. So the increase of pH in these soils is very important when carrying out the revegetation, and biochar could be used as a liming amendment for enhancing nutrient availability and plant survival during the early stages of vegetation establishment productivity

[3,22, 37].

4.2 Evolution of the Total Carbon (TC) at the three depths and over the 11-month period.

As previously mentioned, one of the most important problems when recovering mine soils is their low organic matter content [4]; we also observed this low TC content in the settling pond soil. As a result, we applied the different treatments in an attempt to increase it. After applying the different treatments to soil S, the TC increased considerably. This increase of carbon content in the soil is very important because according to authors such as [39] carbon is an indicator associated with soil quality, fertility and health.

At depth 0-15cm we saw the same behaviour as with the pH, in other words, in the settling soil where we applied the treatments STB and STBP, there was a greater increase in the TC. This is not only due to contribution of TC provided by the elements used to make the technosol, such as solid urban waste, sewage sludge and remnants from agri-food industries [40-44], but also from

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Anexo I the carbon provided by the biochar, as previously demonstrated by [16,17]. Fowles [14] already showed a better behavior of the amendments when they are combined with the biochar. The carbon contribution is important for the recovery of degraded soils, as the increase in organic carbon in the soil increases its possibility to retain the water that is available, increases the concentration of nutrients in bioavailable form, and improves the structure of the soil and other physical properties [46]. At depths 15-30cm and 30-45cm, the increase in TC after applying the different treatments was very clear, especially at 11 months. This shows that these treatments increase the TC at depth, and not only in the first few centimetres of the soil, in the same way as the pH. The comparison between treatment at these depths (15-30cm and 30-45cm) at the end of the time the treatment without biochar STP presented the highest contents of TC. This may be due to the carbon being fixed in the superficial depth in the treatments elaborated with technosol and biochar. On the other hand, the higher carbon content in STP compared to ST may be due to the action of the roots of plants.

4.3 Evolution of the Total Nitrogen (TN) at the three depths and over the 11-month period.

Settling pond soil has undetectable amounts of TN, which is a significant problem when attempting to grow stable plant cover on its surface. The inflow of nitrogen is very low in a mine soil compared to forested or agricultural land [47]. At depths 15-30cm and 30-45cm, the contents were low (below TN <0.1%), possibly due to the lower effect of the treatments both in terms of their capacity to provide TN and their retention, as the nitrogen is lost quickly by leaching. After applying the different treatments, it was not possible to increase the TN content.

This increase is important, as the availability of N to plants is a universally important aspect of soil quality, and nitrogen often represents an immediate limitation to plants [48].

The treatment that raised the nitrogen content the most was the one made with technosol and which had Brassica Juncea L. cultivated on it (STP). The fact that this treatment increased the nitrogen content more than the treatments that combined technosol and biochar (STB,

STBP) is due to several factors; the first is that this treatment (STP) was made of 15% technosol, containing an average of 17.6±0.50 mg/kg of N. In turn, the treatments that combined

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Anexo I technosol and biochar were made of 11% technosol and 4 % biochar (STB, STBP), which means they contained a lower proportion of technosol and included biochar, which contained

5.34±0.22mg/kg of nitrogen. In this case, the biochar has a diluting effect in terms of the nitrogen content. The higher nitrogen content in the technosol is due to the fact that it was made using purification plant sludge and remnants from agri-food industries, which are rich in nitrogen [44, 49,50].

In turn, the fact that the treatment with compost and Brassica Juncea L. (STP) had a higher N content in comparison with the treatment only using compost (ST) at the end of the experiment is probably dueto the effect of the Brassicas on the rhizosphere. Root exudates are important factors that structure the bacterial community of the rhizosphere. It is established that seed/root exudates of plants can be processed as nutrients leading to enhanced growth and a higher prevalence of degrading strains of bacteria [51]. Some non-symbiont nitrogen-fixing bacterial communities are usually associated with certain species of Brassicas; this proliferation of bacteria is the result of various exudates released by the root [52,53]. Due to these factors,

Brassica juncea L. is capable of providing significant amounts of nitrogen to the soil, as demonstrated by [54].

-1 4.4 Evolution of the cation exchange capacity (CEC) (cmol(+)kg ), base saturation

(V%), and aluminium saturation (Al%) at the three depths over the 11-month period.

As previously discussed, at depth 0-15cm, both controls had a low CEC, especially the neutral control (SS). This is a major obstacle to recovering a degraded soil, as a low CEC means the soil has a low resistance to changes in soil chemistry that are caused by land use [55]. At depth 0-15cm, all of the treatments were able to increase the CEC, although as time passed the treatments that combined technosol and biochar (STB, STBP) had higher values. These results agree with those found by authors such as [56] who showed that the application of amendments made with residues increases the cation exchange capacity. On the other hand, [1] have already shown that amended soils benefit from the large, oxidized surface area of biochar and its porous structure. Soils amended with biochar have an increased soil charge density (potential cation

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Anexo I exchange capacity, CEC, per unit surface area) in comparison to non-amended soils. According to [57] the retention of nutrients in terms of cation exchange capacity increase when applying biochar to the soil. This is important, as the cation exchange capacity is a major controlling agent of nutrient availability for plant growth, soil pH, and the soil reaction to fertilisers and other ameliorants [55]. If we break down the CEC at depth 0-15cm, we can see that the base saturation in the treatments (V%) is 99%, but that the aluminium saturation (Al%) is 0%, indicating that the binding sites of the soil in these treatments are saturated with Ca2+, K+, Mg2+

Na+. This saturation of basic cations is important when it comes to increasing the pH, but also because they are nutrients for plants. The treatments we applied that combined technosol and biochar (STB, STBP) had a higher CEC in the final months, due to the higher cation retention capacity of the biochar, as demonstrated by [36,55], as the biochar has a high aromaticity, high surface area, and a negative charge. At depth 15-30cm and all at of the times, the treatments applied, while not always having a higher CEC, did always have a higher base saturation (V%) and lower aluminium saturation (Al%). This indicates that at this depth, the treatments that were applied were still effective. At depth 30-45cm at 3 and 7 months, this trend was maintained, although in time three the V% decreased and the Al% increased in the treatments. This may be due to a decreased effectiveness of the treatments, or that they still needed more time to act. At depths 15-30cm and 30-45cmin 3 and 7 months, treatments STB and STBP had a higher CEC and base saturation, once again clearly revealing the positive effect of biochar in combination with technosol [1, 14].

4.5 Evolution of nutrients at the three depths and over the 11-month period.

At all of the depths and in all of the times studied, it was found that in general, all of the treatments increased the contents of macronutrients such as Ca, Na and K. The application of soil amendments improves soil nutrient content. This is due to the increase of organic matter which improves the EC and it has an effect on the increase of nutrient contents [56]. The nutrient content at depth 0-15cm was higher in the soil amended with the different treatments, due to the contribution made by the elements used to make the technosol and biochar. The

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Anexo I contribution of nutrients by technosols to soils degraded by activities such as mining has already been demonstrated by [10]. Biochar contributes a smaller amount of nutrients at first, although because of its high capacity to retain nutrients, as time passed it retains the nutrients provided by the technosol, and releases them slowly [45]. This is why the nutrient content at 7 and 11 months was generally higher in treatments STB and STBP. As a result, the benefits of applying biochar to these highly weathered systems include the prevention of nutrient loss by leaching, and the retention of nutrients in the root zone [57].

This retention of nutrients is clearer at depth 15-30cm at 3 and 7 months, in which the treatments combining technosol and biochar (STB, STBP) generally had higher contents of the nutrients studied, while at 11 months this effect was no longer visible. Depth 30-45cm, the retaining effect of the biochar was observed at 11 months. It is possible that this retaining effect of the biochar did not become apparent until at 11 months at depths 15-30cm and 30-45 cm due to the fact that the biochar had still not taken effect at depth in a constant manner, perhaps because the acidic pH and lack of organic matter in the final depths did not allow it to act within the timescale of the experiment.

4.6 Limiting factors for plant production at depth 0-15cm.

With regard to the limiting factors for plant production, we will focus on depth 0-15 cm because is the root range zone and discuss the most interesting aspects. The mine soil was affected by the c Factor due to its low pH, which is a limiting factor for the growth of plant life, as most plants and trees cannot grow in a soil with such a low pH. However, the pH that was achieved once the treatments were applied, especially those that combined technosol and biochar (STB, STBP), was an optimum pH for the growth of the majority of plants. Both the mine soil and the neutral control (SS) were affected by the Ca Factor, possibly due to the fact that the low pH of S and SS reduces the availability of Ca to plants. However, the pH range of the soil after treatment is optimal for both the solubility and bioavailability of Ca for plants [55].

Despite the fact that according to [29, 30] the settling pond soil would only be affected by the e

Factor in time 1, several authors have demonstrated that soils with a CEC with values of less

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Anexo I than 6 are considered as very low, meaning that the soil has a low resistance to changes in soil chemistry that are caused by land use. This is one of the reasons why vegetation is not able to establish in settling pond soil without being treated [55]. With regard to K, both S and SS were affected by the k Factor throughout the entire duration of the experiment, and its values are critical for plant growth. After applying the different treatments, the critical values for K were exceeded. Although it was found to be a nutrient whose concentration decreased over time in all of the treatments, despite this decrease the values were always found to be well above the value

-1 of 0.2 cmol(+)kg considered to be critical [29, 30]. Due to the starting material of the settling pond soil, and in the case of the treatments because of the elements used to make them, none of them were affected by the Mg Factor. Only the neutral control SS had a deficit of Mg, possibly due to its origin and the processing of the sand. The settling pond soil and the neutral control (S,

SS) were seriously affected by the N Factor as their TN content was not detectable. None of the treatments that were applied were affected by this factor. This increase in the nitrogen content is significant, as it is a highly important aspect in recovering the quality of a soil. It is also a nutrient that plays a crucial role in plant production, as when it does not reach a given value, it becomes a limiting factor for plant production [48].

4.7 Harvested biomass of Brassica juncea L.

Brassica juncea L planted in the settling pond soil (S) and in the control soil (SS) did not grow over time. These soils showed deficits in certain factors, such as low nutrient content and low pH. This difficulty in establishing stable plant cover in mine soils has already been studied by [7], who states that degraded mine soils are a man-made habitat which experience a wide range of problems for establishing and maintaining vegetation. The plants grew without any problems throughout the whole of the experimental period both in the treatment made only using technosol (STP) and in the treatment using technosol and biochar (STBP). The fact that the plants grew once the different treatments were applied to the soil was also demonstrated by

[7], who states that the use of organic amendments to improve the soil helps to consolidate vegetation in settling pond soils. However, the STPB treatment proved to be more effective than

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STP. In the STPB treatment, the Brassicas had a larger biomass, which increased over time. The larger biomass that was harvested is possibly due to the characteristics the biochar provides to this treatment. Biochar is capable of reducing the leaching of nitrates, allowing plants to use nutrients more efficiently, increasing the pH of the soil, and improving its structure [58]. The nutrients provided by the technosol and the retention capacity of the same by the biochar are a good combination for the vegetal growth. Apart from the larger biomass found in the plants growing in STBP, it was found that the plants in this treatment were higher, indicating that they grew more vigorously. The good development of Brassica juncea L. plants is important because vegetation improves the soil physical, chemical, and biological condition of mine soils [39].

4.8 Principal component analysis (PCA) of the soil samples

The concentrations of the nutrients analysed at depth 0-15cm were selected to perform a principal component analysis (PCA) (Table 6). The two principal components obtained accounted for 97 % of the total variance, according to the position of the soil samples in the scatter plot (Fig.7). In the case of the principal component analysis (PCA) in the soil samples we focused on the evolution of these components in the first depth over time, as this had the greatest influence on the plants used in this experiment.

At 3 months, the treated soils significantly changed their nutrient concentration in comparison to the controls (S and SS). The component score coefficients matrix obtained (Table

6) showed that S and SS are not influenced by any of the nutrient contents. Nevertheless, the soils that were only treated with technosol (ST, STP) were positively influenced by the concentration of most of the nutrients that were analysed (Ca, K, Mg, Mn, Na), while the soils treated with technosol+biochar (STB, STPB) were positively influenced by the concentrations of Mg, Mn, and Na (Figure 7). The scatter plot shows that treated soils were negatively influenced by Fe. (Table 6, Figure 7).

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Fig. 7 Scatter plot with the two principal components obtained in the PCA (PC 1 and PC 2) in height 1

At 7 months, the nutrient concentration remained higher in the treated soils. However, in this case the soils treated with technosol and biochar were also positively influenced by the concentration of Ca and K, as well as by the concentration of Mg and Na, while the soil that was only treated with technosol behaved in the same way as in time 1 (Table 6, Figure 7). Finally, at

11 months (Table 6, Figure 7), the treated soils significantly changed their nutrient concentration in comparison to S and SS, continuing to be positively influenced by the concentration of most of the nutrients that were analysed in the same way as in time 2.

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Table 6. The component score coefficients matrix from the PCA depth 0-15cm Indicators PC1 PC2 Ca 0.57 0.77

Fe -0.11 -0.91 K 0.58 0.75 Mg 0.73 0.66

3 months Mn 0.89 0.16 Na 0.87 0.35 Ca 0.86 0.41

Fe -0.56 -0.44 K 0.97 0.04 Mg 0.94 0.24

7 months Mn 0.06 0.96 Na 0.83 0.03 Ca 0.95 0.25 Fe -0.78 0.06 K 0.81 0.38 Mg 0.85 0.37 Mn 0.15 0.95

11 months Na 0.82 0.52

5 Conclusions

The application of designed treatments improved soil quality. The treatment developed with the combination of technosol and biochar showed in general the best results over the experimental time. The increase in pH values occurred at all depths but the highest increase was at depth 0-15cm. Once the treatments were applied the improvement of the TC content was maintained throughout the experimental time. In addition, this increase occurred in the three depths studied. The comparison between treatment at depths 15-30cm and 30-45cm at the end of the time the treatment STP presented the highest contents of TC. This may be due to the carbon being fixed in the superficial depth in the treatments STB and STBP. After treatment, the settling pond soil in depth 0-15cm experienced a considerable increase of its TN, with the highest values in STP. The CEC increased in nearly all of the cases once the settling pond soil had been treated: the CEC was highest in STBP. After analysing the CEC, it was found that the base saturation was higher in the settling pond soil where these treatments were applied, and that also the aluminium saturation was lower in the settling pond soil where these treatments

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Anexo I were applied, especially in STB. This improvement of the CEC is due to the characteristics of the biochar. The high capacity of biochar to retain nutrients was crucial for the treatment made with technosol and biochar to present the highest nutrient contents. Once the settling pond soil was treated, the critical values for the limiting factors for plant production were exceeded. These factors were exceeded to a greater extent in the treatments that combined technosol and biochar.

Finally, the principal component analysis (PCA) in the samples confirmed the effectiveness of the treatments in increasing the nutrient content of the settling pond soil. The capacity of the biochar to improve the settling pond soil was clearly indicated on analysing the biomass of the

Brassica Juncea L., as the biomass and vigour of the plants was greater in the treatments that combined technosol and biochar.

Acknowledgements

This work was supported by the Spanish Ministry of Economy and Competitiveness under project CGL2016-78660-R.

6. References

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CH4, N2O and NH3 emissions during aerobic composting of a chicken manure and high C/N waste mixture. Waste. Manage. 31, 33–38 (2011) - 45. IBI: International Biochar Initiative. http://www.biochar-international.org/. (2015) - 46. Lal, R: Enhancing crop yields in the developing countries through restoration of the soil organic carbon pool in agricultural lands. Land Degrad. Dev. 17, 197–209 (2006) - 47. Ahirwal, J., Maiti, S.K., Satyanarayana Reddy, M: Development of carbon, nitrogen and phosphate stocks of reclaimed coal mine soil within 8 years after forestation with Prosopis juliflora (Sw.) Dc. Catena. 156, 42-50 (2017) - 48. Christensen, B.T: Tightening the Nitrogen Cycle. In: Schjonning P, Elmholt S, Christensen BT (eds) Managing Soil Quality, challenges in modern agriculture. CABI Publishing. London, UK. 44-66 (2004) - 49. Yang, S.X., Liao, B., Yang, Z.X., Chai, L.Y., Li, J.T: Revegetation of extremely acid mine soils based on aided phytostabilization: A case study from southern China. Sci. Total Environ. 562, 427–434 (2016) - 50. Tejada, M., Parrado, J., Hernández, T., García, C: The biochemical response to different Cr and Cd concentrations in soils amended with organic wastes. J. Hazard. Mater. 185, 204–211 (2010) - 51. Kumar, R., Bhatia, R., Kukreja, K., Behl, R.K., Dudeja, S.S., Narula, N: Establishment of Azotobacter on plant roots: chemotactic response, development and analysis of root exudates of cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.). Journal of J. Basic Microbiol. 47, 436–439 (2007) - 52. Germida, J.J., Siciliano, S.D., de Freitas, J.R., Seib, A.M: Diversity of root- associated bacteria associated with field-grown canola (Brassica napus L.) and wheat (Triticum aestivum L.). FEMS Microbiol. Ecol. 26, 43-50 (1998) - 53. Misko, A.L., Germida, J.J: Taxonomic and functional diversity of pseudomonads isolated from the roots of field-grown canola. FEMS Microbiol. Ecol. 42, 399-407 (2002) - 54. Zhou, X., Wu, H., Koetz, E., Xu, Z., Chen, Ch: Soil labile carbon and nitrogen pools and microbial metabolic diversity under winter crops in an arid environment. Appl. Soil Ecol. 53, 49–55 (2012) - 55. Hazelton, P., Murphy, B: Interpreting soil test results. What do all the numbers mean? CSIRO Publishing. Australia. (2007) - 56. Perez-Esteban, J., Escolastico, C., Masaguer, A., Moliner, A: Effects of sheep and horse manure and pine bark amendments on metal distribution and chemical properties of contaminated mine soils. Eur. J. Soil Sci. 63, 733-742 (2012) - Fellet, G., Marchiol, L., Delle Vedove, G., Peressotti, A: Application of biochar on mine tailings: Effects and perspectives for land reclamation. Chemosphere. 83, 1262- 1267 (2011) - 57. Ippolito, J.A., Laird, D.A., Busscher, W.J: Environmental Benefits of Biochar. J Environ Qual. doi:10.2134/jeq2012.0151 (2012) - 58. Beesley, L., Marmiroli, M., Pagano, L., Pigoni, V., Fellet, G., Fresno, T., Vamerali, T., Bandiera, M., Marmiroli, N: Biochar addition to an arsenic contaminated soil increases arsenic concentrations in the pore water but reduces uptake to tomato plants (Solanum lycopersicum L.). Sci. Total Environ. 454-603 (2013)

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6.2. Anexo II. Comparison of the effects of compost versus compost and biochar on the recovery of a mine soil by improving the nutrient content

Anexo II

Comparison of the effects of compost versus compost and biochar on the recovery of a mine soil by improving the nutrient content (Journal of Geochemical Exploration, under review)

Rubén Forján*, Alfonso Rodríguez-Vila, Emma F. Covelo a Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, Lagoas,

Marcosende, 36310 Vigo, Pontevedra, Spain

*Corresponding author, Tel.: +34 986812630; fax: +34 986812556. E-mail: [email protected]

Abstract

A large number of studies on the recovery of mine soils focus on the problem caused by metals, and do not explore in depth the issue of nutrients and vegetation. The creation of stable vegetation is important, as the satisfactory recovery of a degraded soil requires permanent plant cover. Compost is one of the amendments that is most widely used for recovering degraded soils, although once it has been applied to the soil it may quickly lose its nutrients and other properties. Today, compost is combined with biochar in order to enhance the positive effects of compost. In this experiment, we studied the capacity of biochar to increase the nutrient supply, pH, total carbon, total nitrogen and cation exchange capacity caused by the compost. We compared treatments made using compost with others made using compost and biochar. We also attempted to establish a continuous crop of Brassica juncea L. and to verify which treatment resulted in the best development. In order to study the effects of the treatments as fully as possible, the study was carried out at three different depths over the 45-centimetre length of each cylinder. The study lasted a total of 11 months, using a settling pond from a depleted copper mine in Touro (Galicia, north-west Spain). The results of this experiment revealed that the treatments applied increased the pH, nutrient, total carbon and total nitrogen values, and the cation exchange capacity. In general terms, the most effective treatment consisted of the one using compost + biochar followed by planting with Brassica Juncea L. with only some exceptions towards the end of the experimentation period. The ability of biochar

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Key words: Compost, Biochar, nutrients, mine soil, soil recovering, Brassica juncea L.

1. Introduction

The majority of studies on the recovery of mine soils only focus on the problems caused by metals, without exploring the issues of nutrients and vegetation. However, the satisfactory rehabilitation of mine soils requires a permanent plant cover that prevents soil erosion, allowing for the long-term, sustainable development of the soil (Juwarkar et al., 2009). Also, the vegetation can return a large proportion of percolating water to the atmosphere through transpiration, thereby reducing the concentrations of soluble metals entering watercourses. Plant cover also goes a long way towards reducing the visual scars in the landscape caused by large- scale mining operations. Successful revegetation may allow for the recreational use of the land, and even agriculture or forestry if conditions are favourable (Tordoff et al., 2000). The problem with mine soils in terms of supporting vegetation is that they often have poor conditions for plant growth, including poor physical structure, sandy texture, acidity, poor cation exchange capacity (CEC) and low organic matter and nutrient contents, which limit the establishment of vegetation and intensify erosion caused by rain and wind (Perez-Esteban et al., 2012).

In order to increase the content of nutrients in a phytoavailable form in mine soils, a large amount of amendment material has to be used in order to provide organic matter and slowly release nutrients. For this reason, organic wastes have been widely used as amendments in mine reclamation, as they are inexpensive and produced in large quantities (Asensio et al.,

2014). In recent years, there has been strong interest in recycling urban waste by composting and using it as a soil amendment (Paetsch et al., 2016). Compost made using different types of manure provides significant benefits when applied to soil, as the organic matter from manure

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Anexo II acts as a nutrient pool, improves nutrient cycling, increases the CEC and buffer capacity, reduces compaction, and improves physical soil properties such as aggregation, friability, density, root penetration, water retention capacity and water infiltration (Walker et al., 2004).

Several studies, such as Luna et al., (2016) have demonstrated the effectiveness of compost in improving aspects such as the carbon, nitrogen or potassium content. Another amendment currently in use to improve the conditions of degraded soils is biochar. Biochars are biological residues combusted under low oxygen conditions, resulting in a porous, low density, carbon rich material, with a large surface area. When biochar is used as a soil amendment, it has been reported to boost soil fertility and improve soil quality, resulting in increased crop yields. Soil benefits include raising the soil pH, increasing moisture holding capacity, attracting more beneficial fungi and microbes, improving cation exchange capacity (CEC), and retaining nutrients. Given that biochar is highly recalcitrant, the effects of its application may be prolonged over a long period of time. (Beesley et al., 2011, Fellet et al., 2011, Filiberto and

Gaunt, 2013). This ability to retain nutrients indicates that biochar could be used in combination with other amendments in or order to retain the nutrients they provide, as is the case with compost, as one of the problems associated with the use of compost is that once it is applied to the soil, it quickly loses the nutrients it contains, and the carbon it provides is not very recalcitrant. For this reason, some organic amendments are combined with biochar, as this can enhance the positive effects of the compost, as demonstrated by Fowles (2007). Biederman and

Harpole (2013) analysed the results of 371 independent studies, while the meta-analysis by

Biederman and Harpole (2013) showed that the addition of biochar to soils resulted in increased above-ground productivity, crop yield, soil microbial biomass, rhizobia nodulation, plant K tissue concentration, soil potassium (K), total soil nitrogen (TN), and total soil carbon (TC) compared with control conditions. The aim of using these amendments is to recover degraded soils, in order to establish an initial vegetation cover. The early stage of vegetation establishment is particularly crucial and requires good site preparation practices in order to maximise the chance of the revegetation process being successful. Site preparation practices

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Anexo II have the potential to modify soil physicochemical properties, thereby influencing nutrient availability for the plants that are established (Reverchon et al., 2015).

The aim of this study was to compare the effectiveness of several different treatments in recovering nutrients, increasing the pH, total carbon, total nitrogen, and the cation exchange capacity in settling pond soil from a mine. The different treatments were made using only compost or with compost and biochar, and were vegetated with Brassica Juncea L. or left unvegetated. In order to be able to compare the different treatments at different depths, in this study we put settling pond soil from a depleted copper mine in 50-cm cylinders in order to reflect as closely as possible the first few centimetres of soil. Another aim of this study was to determine the ability of biochar to fix nutrients and enhance the positive effects of compost, in order to be able to establish a continuous crop of Brassica juncea L. Studies by authors such as

Lombi et al., (1999), do Nascimento et al., (2006), and Rodríguez-Vila et al., (2014) successfully used Brassica Juncea L. in polluted mine soils.

2. Material and Methods

2.1 Soil sampling

The sample zone is located in an old copper mine in Touro, in north western Spain (8º

20' 12.06'' W 42º 52' 46.18'' N) (Fig. 1). The climate in this zone is Atlantic (oceanic) with precipitation reaching 1886 mm per year (with an average of 157 mm per month) and a mean daily temperature of 12.6ºC. The average relative humidity is 77% (AEMET, 2015). In order to carry out the study, one soil and three amendments were selected: the soil chosen came from a settling pond (S) comprised of waste material resulting from the flotation of sulphides during copper processing. The pool has been dry for several years, and is considered to be soil according to the latest version of the FAO (2006). The three amendments were sand, consisting of washed sea sand provided by the company Leboriz S.L.U, and which was used as a neutral control. The compost (C) consisted of horse and rabbit manure mixed with grass cuttings, fruit and seaweed, and was provided by the company Ecocelta Galicia S.L. (Ponteareas, Pontevedra,

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Spain). The biochar (B) used was made from Quercus ilex wood with a pyrolysis temperature of

400 °C for 8 hours, provided by the company PROININSO S.A.

Fig. 1 Location of the sampled area in Touro mine.

2.2 Greenhouse experiment.

The greenhouse experiment was carried out in cylinders with the aim of reflecting the top 45 centimetres of soil; the cylinders were made of PVC, with a height of 50 cm and a diameter of

10 cm. A porous mesh was placed inside the cylinders, and the settling pond soil was poured

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Anexo II inside. The mesh used for the settling pond soil was not in contact with the PVC for a long period of time (Fig. 2). The cylinders were filled with settling pond soil (S, negative control) settling pond soil and sand (SS, neutral control), and the following treatments:

- Settling pond soil + compost (SC)

- Settling pond soil + compost + vegetated with Brassica juncea L. (SCP)

- Settling pond soil + compost + biochar (SCB)

- Settling pond soil+ compost + biochar + vegetated with Brassica juncea L. (SCBP).

The amendment ratios used are detailed in Table 1. The total weight of each cylinder was

3.5 kg.

Table 1. Proportions used to make the controls and the different treatments

Soil Sand Compost Biochar

S 100%

SS 85% 15%

SC 85% 15%

SCP 85% 15%

SCB 85% 11% 4%

SCBP 85% 11% 4%

The experiment was carried out over an 11-month period at a controlled temperature and humidity (temperature of 22±2 °C, and 65±5 % relative air humidity). A total of 90 cylinders,

(15 cylinders per treatment) were prepared and randomly distributed (S, SS, SC, SCP, SCB,

SCBP). Three cylinders of each type were withdrawn at 3 different times: Time 1= 3 months,

Time 2= 7 months, and Time 3= 11 months. The meshes were removed from the cylinders and processed for analysis 3 different depths: the first from 0-15 cm, the second from 15-30 cm, and

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Figure 2. Cylinder design and the different depths.

2.3 Soil analysis

(TN) were determined in a LECO CN-2000 module using solid samples. Exchangeable cations were extracted with 0.1 M BaCl2 (Hendershot and Duquett, 1986) and their concentrations were determined by ICP-OES (Optima 4300 DV; Perkin-Elmer). Pseudototal metal contents were extracted with aqua regia by acid digestion in a microwave oven (Milestone ETHOS 1, Italy).

Metal concentrations were determined by ICP-AES (Optima 4300 DV; Perkin-Elmer). A series of critical values were assigned to each of the chemical parameters, based on the model of the

SFCC (Soil Fertility Capability Classification) proposed by Buol et al., (1975) and adapted by

Macías and Calvo (1983), Calvo de Anta and Macías (1987), and Calvo et al., (1987). These were used to evaluate the limiting factors for plant production.

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2.4 Plant growth and determination of metals in plant tissues

The Brassica juncea L. plants were pre-germinated in seedbeds until they grew two fully expanded leaves, and were then transferred to the cylinders (SCP, SCBP). The plants were harvested in the same state of maturity, for comparison in the same state of development in the three times (Time 1= 3 months, Time 2= 7 months, Time 3= 11 months). Growth was allowed under greenhouse-controlled conditions, with a photoperiod of 11:13 light/dark, a temperature of 22±2 °C and 65±5 % relative air humidity. At the end of each time period, the height of the plants was measured, and they were carefully washed with deionised water. Fresh biomass was weighed immediately, and dry mass was assessed after oven drying for 48 hours at 80 °C and cooling at room temperature. The plant tissues, divided into roots and shoots, were air dried and ground.

2.5 Statistical analysis

All of the analytical determinations were performed in triplicate. The data obtained were statistically treated using the programme SPSS version 19.0 for Windows. Analysis of variance (ANOVA) and test of homogeneity of variance were carried out. In case of homogeneity, a post hoc least significant difference (LSD) test was carried out. If there was no homogeneity, Dunnett’s T3 test was performed. Principal component analysis (PCA) was also carried out. Student’s T-test was using to compare the results for the biomass and the height between SCP and SCBP.

3. Results

3.1 General characteristics of the settling pond soil (S), sand (SS), compost (C), and biochar (B).

The biochar and compost had higher pH values than the controls (S and SS), and the soil from the settling pond (S) had an acidic pH (P < 0.05) (Table 2). Total carbon (TC) was significantly higher in the biochar compared to the compost (C), soil from the settling pond and sand (P < 0.05) (Table 2). The compost had the highest total nitrogen content (TN) (P < 0.05)

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(Table 2). The TN values were undetectable in S and were extremely low in the sand (Table 2).

S had the highest Fe and Mn concentrations (Table 2).

Table 2. Characteristics of the mine tailing (S), sand (SS), compost (C) and biochar (B).

S SS C B

pH 2.73±0.07d 3.83±0.55c 6.47±0.02b 9.90±0.02a . -1 T.C g kg 1.93±0.15c 2.76±0.60c 276±2.51b 676±4.58a . -1 T.N mg kg u.d 0.10±0.01c 21.3±1.02a 5.34±0.22b

Ca 13.3±0.02c 9.65±0.05c 6455±153a 531±4.88b

K mg.kg-1 6.40±0.89c u.d 3041±46.53b 3243±23.1a Mg 216±2.10c 1.83d 1038±14.9a 548±11.6b Na 27.4±0.90c 7.95d 987±12.4a 65.4±0.07b 3+ Al 2.32±0.02a 0.02b u.d u.d

2+ Ca -1 cmol(+)kg 0.07c 0.05d 32.2±0.76a 2.66±0.02b + K 0.02c u.d 7.78±0.11b 8.30±0.05a 2+ Mg 1.78±0.01c 0.01d 8.54±0.12a 4.51±0.09b 2+ Na 0.14c 0.04d 4.93±0.06a 0.34b -1 CEC cmol(+)kg 4.35c 0.14d 53.5±1.07a 15.8±0.17b Cu 437±1.74a 46.6±0.96c 193±1.14b 27.2±1.29d

Pb Pseudototal 17.7±0.41b 10.1±0.27c 26.6±0.96a u.d (mg.kg-1) Ni 10.8±0.97c 8.41±0.52c 49.7±1.71a 25.3±0.79b Zn 64.7±1.28b 18.7±0.85c 403±3.33a 62.4±1.70b

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level. Typical deviation is represented by ±.

The compost had the highest concentrations of Ca, Mg and Na (Table 2). K concentrations were significantly higher in the biochar compared to S, SS and C (P < 0.05)

(Table 2). In relation to the cation exchange, the Al3+ content in S was significantly higher than that in the sand, compost and biochar (P < 0.05) (Table 2). The compost (C) had higher concentrations of Ca2+, Mg2+ and Na+ than S, SS and B. The Na2+ content in B was significantly higher than that in the sand, compost and biochar (P < 0.05) (Table 2). The exchangeable cation capacity (CEC) of C was significantly higher than in the controls (S and SS) and biochar (B).

The pseudototal concentration of Cu in the settling pond soil was higher than in C and B (P <

0.05) (Table 2). The compost had the highest pseudototal concentration of Pb, Ni and Zn (Table

2).

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3.2 Evolution of the pH at the three depths and over the 11-month period.

In depth 0-15 cm at time 1, all of the treatment had higher pH values than the controls S and SS. The SCB and SCBP treatments (settling pond soil treated with compost and biochar) had the highest pH at times 2 and 3 (Fig. 3A).

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05. Error bars represent typical deviation

Figure 3. Evolution of the pH at three depths and over the 11-month period.

In depth 15-30 cm at time 1, the SC and SCB treatments and the neutral control (SS) had a significantly higher pH than the untreated settling pond soil (P < 0.05) (Fig. 3B). At time

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2, SCB had the highest pH, and at time 3 SCBP had the highest pH (Fig. 3B). At depth 30-45 cm at time 1, the controls S and SS had the highest pH values. At time 2, the neutral control

(SS) and the SCP and SCBP treatments had the highest pH values. The treatment made using compost and biochar and vegetated with Brassica Juncea L. (SCBP) had the highest pH values in time 3 (P < 0.05) (Fig. 3C).

3.3 Evolution of Total Carbon (TC) at the three depths and over the 11-month period.

In depths 0-15 cm and 30-45 cm, treatments SCB and SCBP had the highest TC concentration over time (Fig.4A, 4C).

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05. Error bars represent typical deviation

Figure 4. Evolution of Total Carbon (TC) at the three depths and over the 11-month period

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In depth 15-30 cm, at time 1, the SCB treatment (settling pond soil treated with compost and biochar) had a significantly higher TC concentration than the untreated settling pond soil (P

< 0.05) (Fig. 4B). At time 2, SC and SCBP had the highest TC concentrations, although at time

3, the SCBP treatment (settling pond soil treated with technosol compost + biochar + Brassica

Juncea L.) had the highest TC concentrations (Fig. 4B).

3.4. Evolution of Total Nitrogen (TN) at the three depths and over the 11-month period.

The TN was only detected at deptht 0-15 cm and only in the treated settling pond soil.

At time 1 the settling pond soil that was only treated with compost (SC) had the highest TN concentration (Fig. 5). At times 2 and 3, the SCP treatment (settling pond soil treated with compost + Brassica Juncea L.) had the highest TN concentration (P < 0.05) (Fig. 5).

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05. Error bars represent typical deviation

Figure 5. Evolution of Total Nitrogen (TN) at depth 0-15 cm and over the 11-month period

3.5. Evolution of the cation exchange capacity (CEC), base saturation (%), and aluminium saturation (%) at the three depths and over the 11-month period.

In depth 0-15 cm, the settling pond soil treated with compost+biochar and Brassica

Juncea L. (SCBP) had the highest CEC at time 1-2, but at time 3, SCP had the highest CEC (P <

0.05) (Table 3). At time 1, the base saturation (V%) was higher in the treated settling pond soil

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(SC, SCP, SCB, SCBP) than in the controls (S, SS) (Table 3). The settling pond (S) and the neutral control (SS) had a higher aluminium saturation (Al%) than the treated settling pond soil

(SC, SCP, SCB, SCBP) (Table 3).

Table 3. Evolution of the cation exchange capacity (CEC), base saturation (V%), and aluminium saturation (Al%) at three depths and over the 11-month period. S SS SC SCP SCB SCBP

CEC 0.84±0.05d 0.94d 21.5±2.30c 36.5±0.77b 23.4±1.45c 41.0±2.06a

V% 35.1 27.7 100 100 100 100 Time 1 Time Al% 64.9 72.3 0 0 0 0

CEC 3.68±0.04e 5.05d 25.4±0.40c 28.4±1.28c 31.0±1.17bc 34.7±1.07a

15 cm 15

- V% 36.7 36.1 99.9 100 100 100

Time 2 Time Al% 63.3 63.9 0.01 0 0 0

Depth 0 Depth

CEC 10.7±0.47c 5.86d 30.0±1.79ab 39.3±0.08a 29.1±0.96b 29.9±0.98b

V% 43.3 41.8 99.9 100 100 100

Time 3 Time Al% 56.7 58.2 0.01 0 0 0

CEC 0.94±0.02c 1.12±0.01c 1.32±0.11c 2.91±0.15b 2.85±0.08b 10.4±0.33a V% 27.3 25.4 76.6 92.1 62.9 48.9

Time 1 Time Al% 72.7 74.6 23.4 7.90 37.1 51.1

CEC 3.06±0.49c 4.12±0.84b 3.46±0.07bc 2.74±0.25c 4.43±0.06b 6.75±1.86a

30 30 cm

- V% 36.8 37.7 43.6 56.5 48.0 44.1

Time 2 Time Al% 63.2 62.3 56.4 43.5 52.0 55.9

Depth 15 Depth CEC 10.6±0.12a 11.0±0.13a 3.14±0.04b 4.18±0.26b 1.19±0.17c 3.39±0.39b

V% 40.8 38.3 41.0 62.6 71.4 96.4

Time 3 Time Al% 59.2 61.7 59.0 37.4 28.6 3.56

CEC 1.09±0.01c 1.15±0.10c 1.40±0.27b 2.03±0.10b 8.24±0.41a 10.1±0.76a V% 22.8 41.5 60.0 69.7 34.7 35.0

Time 1 Time Al% 77.2 58.5 40.0 30.3 65.3 65.0

CEC 3.23±0.16b 4.61±0.10b 4.06±0.18b 4.73b 6.11±0.34a 8.16±3.27a

45 45 cm

- V% 38.4 32.8 43.0 47.8 35.5 42.1

Time 2 Time Al% 61.6 67.2 57.0 52.2 64.5 57.9

Depth 30 Depth CEC 15.6±0.47a 11.5±0.01b 3.93±0.12c 2.86±0.18d 0.94e 1.64±0.14e V% 38.1 40.1 40.0 43.2 38.6 43.5

Time 3 Time Al% 61.9 59.9 60.0 56.8 61.4 56.5 For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level. Typical deviation is represented by ±.

In depth 15-30 cm, at times 1-2, SCBP had the highest CEC at time 1-2, while at time 3 the neutral control had the highest CEC (P < 0.05) (Table 3). The treatment using only compost and Brassica Juncea L. (SCP) had the highest base saturation at times 1 and 2, but at time 3 the highest V% was shown by the settling pond soil treated with compost+biochar + Brassica

Juncea L. (SCBP) (Table 3). The control (S, SS) had the highest aluminium saturation (Al%) over time (P < 0.05) (Table 3).

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In depth 30-45 cm, the SCBP treatment had the highest CEC between times 1 and 2, while at time 3, the untreated settling pond soil (S) had the highest CEC (Table 3). The treatment only using compost and Brassica Juncea L. (SCP) had the highest base saturation at times 1 and 2, but at time 3 the highest V% was shown by the settling pond soil treated with compost +biochar + Brassica Juncea L. (SCBP) (Table 3). The settling pond soil had the highest aluminium saturation (Al%) at times 1 and 3, while the neutral control had the highest Al% at time 2 (Table 3).

3.6. Evolution of nutrients at the three depths and over the 11-month period.

In depth 0-15 cm, the neutral control (SS) had the highest Fe concentration at time 1-2, while at time 3 the settling pond soil (S) had the highest Fe concentration (P < 0.05) (Table 4.1).

At time 1, SC had the highest Mn concentration and SCP had the highest Ca concentration. The settling pond soil treated with compost + biochar + Brassica Juncea L. (SCBP) had the highest

K, Mg and Na concentrations (Table 4.1).

Table 4.1. Evolution of nutrients in three depths and along the 11 months

Depth 0- S SS SC SCP SCB SCBP 15 cm Ca 9.51±1.74e 6.53±0.26e 2837±310c 4818±93.1a 2771±161.4d 4641±211.6b

Fe 5.06±0.12b 8.71±0.57a 0.18±0.04c 0.24±0.03c 0.06±0.03d 0.03±0.01d K 9.18±0.07e 7.89±0.41e 593±5632d 1511±37.8b 991±62.7c 2572±146.0a Mg 12.0±2.66d 11.4±0.09d 551±61.4c 662±13.7b 581±35.9c 790±40.7a

Time1 Mn 0.66±0.16d 0.94±0.02d 70.6±5.78a 1.90±0.04c 4.11±0.19b 2.04±0.06c Na 25.7±2.20e 21.7±0.51e 247±21.9d 615±19.9b 458±37.5c 956±60.6a Ca 30.6±0.31c 38.7±1.05c 3690±64.6b 4358±208a 3607±135b 4529±137a

Fe 323±8.97b 696±42.9a 0.92±0.29c 0.08±0.05c 0.06±0.05c u.d K 1.68±0.69d 2.12±0.51d 481±4.56c 699±16.7b 790±32.6b 1262±44.2a Mg 128±1.10e 184±0.57d 612±7.19b 479±21.4c 904±39.9a 768±23.0a Time 2 Mn 4.17±0.07c 4.10±0.14c 54.4±1.62a 0.84d 5.53±0.05b 1.60±0.01d Na 22.3±0.14e 17.8±1.24e 130±1.57d 176±2.43c 701±17.4a 510±16.8b Ca 94.8±4.29c 58.4c 4488±260b 5647±194a 4441±148b 4639±151b

Fe 497±135a 143±3.00b 0.85±0.08c u.d u.d 0.07±0.05c K u.d u.d 500±34.1b 596±18.7a 225±5.42d 432±14.5c Mg 496±24.1b 253±1.63c 698±54.4a 556±16.3b 704±22.8a 614±19.0ab Time 3 Mn 9.89±0.54b 6.82±0.13c 35.9±1.75a 0.59e 2.32±0.15d 0.61±0.05e Na 14.2±0.92c 11.9±0.48c 115±7.60b 125±3.53a 110±3.74b 115±4.96b

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level Typical deviation is represented by ±.

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At time 2, the treated settling pond soil with compost (SC) had the highest Mn concentration.

SCBP had the highest Ca and K concentration, while the highest Mg concentration was in the treatments with compost and biochar (SCB, SCBP). The SCB treatment had the highest K concentrations (P < 0.05) (Table 4.1). At time 3, the settling pond soil treated with compost and

Brassica Juncea L. (SCP) had the highest Ca, K and Na concentrations, and settling pond soil only treated with compost had the highest Mn concentration. The SC and SCB treatments had the highest Mg concentration (Table 4.1).

In depth 15-30 cm, at time 1, SCBP had the highest nutrient concentration, except for

K and Na. SCP had the highest K and Na concentration (P < 0.05) (Table 4.2). At time 2, SCB and SCBP had higher concentrations of Na than the controls (S, SS) and the treatments without biochar (SC, SCP) (Table 4.2).

Table 4.2. Evolution of nutrients in three depths and along the 11 months

Depth S SS SC SCP SCB SCBP

15-30 cm Ca 6.47±0.14e 8.18±0.08e 57.0±5.12d 228±1.26b 138±4.10c 329±40.4a

Fe 5.42±0.01c 6.09±0.32c 3.73±0.36cd 2.35±0.45d 22.7±1.95b 24.6±0.03a K 8.30±0.87e 14.7±0.29d 58.0±6.35c 209±17.1a 58.1±2.24c 130±1.39b Mg 8.64±0.56d 9.18±0.53d 32.9±2.88c 44.6±3.08c 82.3±3.44b 335±17.8a

Time1 Mn 0.77±0.02e 0.96d 2.04±0.15c 0.77±0.01e 3.69±0.10b 10.8±0.30a Na 21.2±1.21d 23.8±1.79d 54.8±3.84c 124±8.98a 50.3±1.42c 70.0±1.81b Ca 28.8±4.60d 45.5±9.96d 72.9±2.65c 105±13.0b 98.6±1.25bc 178±32.4a

Fe 232±57.8b 487±110a 108±0.54e 97.7±15.8e 143±3.21d 175±26.9c K 1.59±0.70c 0.54±0.42c 7.45±0.24b 51.5±2.17a 33.7±4.56a 10.1±5.41b Mg 103±18.6c 148±32.2b 124±0.80bc 88.4±8.13c 152±1.38b 218±50.8a Time 2 Mn 3.46±0.53c 3.64±0.79c 6.74±0.38b 2.63±0.31c 5.57b 10.1±3.55a Na 20.6±0.54bc 16.7±2.33c 16.4±0.81c 28.2±1.57b 53.9±8.62a 47.2±0.50a Ca 110±2.71b 123±7.29b 63.9±6.06c 248±21.5a 115±26.3b 538±78.8a

Fe 109±3.08b 47.70.80d 62.4±0.01c 157±13.3a 9.39±0.29e 1.77±0.35e K u.d u.d 1.22±0.06c 10.7±0.83b u.d 30.3±4.55a Mg 452±4.74a 430±3.45a 108±0.63b 112±8.74b 25.3±2.98d 49.4±3.63c Time 3 Mn 13.1±0.14b 16.1±0.46a 5.32±0.07c 3.68±0.27d 2.52±0.33de 2.15±0.22e Na 10.6±0.13c 11.5±0.58bc 10.7±0.08c 11.8±0.02b 10.0±0.83c 13.6±0.63a

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level Typical deviation is represented by ±.

The highest Ca, Mg and Mn concentrations were shown by SCBP. The SCB treatment had the highest K concentrations, while the neutral control had the highest Fe concentration (P < 0.05)

(Table 4.2). At time 3, S and SS had higher Mg and Mn concentrations than the treatments. The

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SCP and SCBP treatments had the highest Ca concentrations. The SCBP treatment had the highest K and Na concentrations, and the highest Fe concentrations were in SCP (Table 4.2).

In the depth 30-45 cm, at time 1, the SCB and SCBP treatments had the highest Ca, Fe,

Mg and Mn concentrations. The highest K and Na concentrations were shown by SCBP (P <

0.05) (Table 4.3). At time 2, the SCBP had higher Ca, Mg and Na concentrations than the other treated settling pond soils and controls. SCB and SCBP had the highest Mn concentrations. SS and SCP had the highest Fe and K concentrations respectively (Table 4.3). At time 3, the settling pond soil (S) had higher Ca, Mg and Mn concentrations than the treated settling pond soil and the neutral control. SS and SCP had the highest Fe concentrations, and SC had higher

Na concentrations than the controls and the other treated settling pond soil (P < 0.05) (Table

4.3).

Table 4.3. Evolution of nutrients in three depths and along the 11 months

Depth S SS SC SCP SCB SCBP

15-30 cm Ca 7.01±0.18d 5.38±0.75d 38.0±9.54c 80.4±6.50bc 118±0.42a 113±11.3a Fe 6.74±0.52c 4.65±0.82c 3.16±0.56c 3.26±0.19c 300±3.18b 412±24.1a

K 7.50±0.19d 7.26±0.40d 40.4±6.05bc 109±4.59a 63.1±2.30b 19.6±1.80c Mg 13.9±0.03d 6.72±2.12d 31.6±5.90cd 32.0±1.91c 231±9.13b 343±23.3a

Time1 Mn 0.48±0.01c 0.63±0.20c 1.97±0.33b 1.24±0.11b 12.7±0.41a 11.4±0.85a Na 20.8±0.62d 23.1±2.70d 54.2±11.3bc 90.1±2.36a 33.3±1.11c 13.8±1.79e Ca 55.4±1.79c 54.1±10.1c 74.5±1.25b 95.7±0.39b 75.9±0.47b 159±33.1a Fe 266±9.82c 830±34.5a 363±13.2b 397±4.08b 276±15.1c 218±91.0c

K 1.00±0.37c 0.27±0.17c 2.87±1.37c 71.1a 14.1±1.07b 9.95±3.59bc Mg 102±4.67d 144±4.57c 150±6.26c 165±2.56bc 187±13.3b 288±80.5a

Time 2 Mn 3.51±0.13b 3.42±0.06b 4.07±0.15b 3.73±0.07b 10.9±0.50a 11.8±7.62a Na 19.9±1.36c 6.09±3.10d 21.1±3.49c 42.8±4.71ab 37.5±5.05b 44.6±1.76a Ca 184±5.66a 150±0.26b 32.7±1.59b 34.2±1.63b 16.8±0.32c 32.8±3.59b Fe 77.9±1.81b 218±7.40a 105±3.15ab 115±8.76a 22.6±0.07c 37.4±3.26c

K u.d u.d u.d u.d u.d u.d Mg 606±18.5a 465±0.49b 161±2.85c 109±8.59d 26.6±0.07f 60.0±5.03e

Time 3 Mn 21.3±0.70a 12.7±0.12b 3.99±0.10c 3.22±0.17d 1.73±0.01e 2.26±0.15e Na 10.3±0.56b 11.3±0.19b 12.6±0.25a 10.4±1.07b 8.39±0.11c 7.72±0.45c

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level Typical deviation is represented by ±.

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3.7 Limiting factors for plant production in depth 0-15 cm.

All of the factors and values detailed below are shown in table 5. The high acidity of the settling pond soil, over time, is a limiting factor for plant production (Factor c). The controls (S, SS) had deficient amounts of Ca that gave rise to the limiting Factor Ca. The CEC values are below the limit set by the Factor e at time 1 for the controls, and at time 2 only for S. The K values were a limiting factor (Factor K) in S and SS over time. The controls had a deficiency of Mg2+ over time; this resulted in the presence of a limiting factor for plant production (Factor Mg).

Only S at time 1 exceeded the limit set by the Factor n. The settling pond soil and the neural control were severely limited for plant production according to Factor N over time.

Table 5. Limiting factors for crop production linked to the content of bases in the complex change in depth 0-15 cm.

Factor c Factor Ca Factor e Factor K Factor Mg Factor n N

S 3.50 0.05 0.84 0.02 0.08 16.0 0 SS 4.08 0.03 0.94 0.02 0.08 12.5 0

SC 7.07 14.1 21.5 1.52 4.54 5.78 1.96 SCP 6.23 24.0 36.5 3.87 5.45 8.44 1.56

Time 1 SCB 7.20 13.8 23.4 2.54 4.79 9.78 1.76 SCBP 7.03 23.2 41.0 6.58 6.50 11.6 1.80

S 3.02 0.15 3.68 0 1.06 3.30 0 SS 3.81 0.19 5.05 0.01 1.52 1.95 0

SC 5.21 18.46 25.4 1.23 5.04 2.59 1.41 SCP 5.89 21.7 28.4 1.79 3.95 3.13 1.55

Time 2 SCB 7.47 18.04 31.0 2.02 7.44 11.3 1.48 SCBP 6.89 22.6 34.7 3.23 6.32 7.35 1.43 S 2.63 0.47 10.7 0 4.08 0.75 0 SS 3.33 0.29 5.86 0 2.08 1.18 0

SC 5.42 22.4 30.0 1.28 5.74 1.94 1.89 SCP 5.66 32.5 39.3 1.53 4.58 1.61 1.99

Time 3 SCB 7.56 22.2 29.1 0.58 5.79 1.93 1.89 SCBP 7.52 23.2 29.9 1.11 5.06 1.95 1.81

-1 -1 -1 -1 Factor c pH<3.5; Factor Ca<1.5cmol(+)kg ; Factor e ECC <4cmol(+)kg ; Factor K <0.2 cmol(+) kg ; Factor Mg <0.4 cmol(+) kg ; Factor n Na> 15%; N Total content <0.1%

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3.8 Harvested biomass of Brassica juncea L.

The Brassica juncea L. was not capable of growing in the settling pond soil (S) or in the neutral control (SS), and so it is not shown in figures 6A and 6B. The Brassica juncea L. biomass harvested from the SCPB treatment was higher than in SCP over time (Fig. 6A; P <

0.05). The Brassica juncea L. harvested in the SCPB treatment was the highest at time 1, but at time 2-3 the Brassica juncea L. harvested in the SCP treatment was the highest (Fig. 6B; P <

0.05).

Figure 6. Havested biomass and heigth of Brassica Juncea L. over the 11-month period. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represents standar deviation

4. Discussion

4.1 Evolution of the pH at the three depths and over the 11-month period.

As indicated in the results section, in general terms all of the treatments were able to increase the pH level in all of the depths by the end of the experiment. The pH increased the most in the SCBP treatment at the end of the experimental period in each depth. As previously demonstrated by Fowles (2007), this demonstrates the ability of biochar to improve the effect of organic amendments. Also, according to several authors (Karer et al., 2015, Zhang et al., 2013), biochar used separately as an amendment is also able to increase the pH. This increase in the pH is important, as the pH is an important for restoration in degraded soil as soil pH has been reported as being a critical factor controlling revegetation success, and biochar could be used as a liming amendment for enhancing nutrient availability and plant survival during the early stage of vegetation establishment productivity (Reverchon et al., 2015, Shrestha and Lal, 2011; Zhang

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Anexo II et al., 2013). In turn, the low pH of the settling pond soil (S) is due to its source, as sulphide minerals in contact with water produce sulphuric acid (Pataca, 2004), and also because this soil is predominated by waste resulting from the flotation of sulphides during copper processing.

4.2 Evolution of the Total Carbon (TC) at the three depths and over the 11-month period.

As with the pH, in the case of the total carbon (TC) we observed a trend whereby the

TC increased in all of the treatments during the experiment, although at the last time and all of the depths, this increase was greater in the treatments that contained (SCB, SCBP). The increased TC resulting from the addition of compost is due to the components used in its manufacture: as previously demonstrated (Canet et al., 2008, Illera-Vives et al., 2015, Pérez-

Esteban et al., 2012), compost containing horse or rabbit manure or seaweed has a high organic matter content. This contribution of TC was favoured by the biochar used, as it has a high carbon content (676 g/kg), and by the fact that the biochar itself also contributes C. The fact that biochar increases the carbon content of the soil has already been demonstrated by Biederman and Harpole (2013) and Madiba et al., (2016). This is not only of interest because of this increase, but also because this carbon is highly recalcitrant, and contributes towards increasing the concentration of nutrients in bioavailable form (Lal, 2006).

4.3 Evolution of the Total Nitrogen (TN) at the three depths and over the 11-month period.

The TN was undetectable in the two controls (S and SS) in the three depths we studied.

In turn, the TN contents in the treatments in the depth 0-15 cm increased significantly, while in depth 15-30 cm and 30-45 cm the contents were low. The content in depth 15-30 cm and 30-45 cm was lower than TN <0.1%, possibly due to the lesser effect of the treatments in their ability to provide TN and in its retention. The increase caused by the treatments applied to the settling pond soil is important, as the availability of N to plants is a universally important aspect of soil quality, and often nitrogen represents an immediate limitation to plants (Christensen, 2004). Not

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Anexo II all of the treatments were equally effective when increasing the TN, as in the last two times, it increased the most in SCP. The fact that the nitrogen content increased more in this treatment than in those combining compost and biochar is due to several factors. The first is the proportion of the mixture used, as treatment SCP was made using 15% compost with an average of 21.3±1.02 mg/kg of TN, while the treatments combining compost and biochar were made using 11% compost and 4% biochar (SCB, SCBP), which means that they contained a lower proportion of compost and included biochar, with a TN content of 5.34±0.22 mg/kg. In this case, the biochar had a diluting effect on the TN content. Another possible reason why the treatments with biochar had a lower TN content is that the Brassica Juncea L. plants harvested from the treatments with biochar grew more, as can be seen from their biomass (Fig. 6), resulting in a greater consumption of nutrients. In turn, the fact that the treatment with compost and Brassica Juncea L. (SCP) had a higher N content in comparison with the treatment only using compost (SC) is due to the effect of the brassicas on the rhizosphere. Root exudates are important factors that structure the rhizosphere’s bacterial community. These non-symbiont bacterial communities are nitrogen fixers, and are usually associated with certain species of brassicas. (Germida et al., 1998, Misko and Germida, 2002). For this reason, Brassica juncea L. is capable of contributing important amounts of nitrogen to the soil, as demonstrated by Zhou et al., (2012)

-1 4.4 Evolution of the cation exchange capacity (cmol(+)kg ), base saturation (V%), and aluminium saturation (Al%) at the three depths and over the 11-month period

In depth 0-15 cm, all of the treatments applied to the settling pond soil were able to significantly increase the CEC in comparison to the controls. This increase is important, because as previously described by Hazelton and Murphy, (2007), a high CEC is indicative of a greater resistance to the chemical changes a soil may experience from its use. In times 1 and 2, this increase was greater in SCBP, as amended soils benefit from biochar’s large surface area and porous structure. Ohsowski et al., (2012) demonstrated that soils amended with biochar have an increased soil charge density (potential cation exchange capacity, CEC, per unit surface area) in

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Anexo II comparison to non-amended soils. This effect was not noted in time 3, in which the treatments without biochar (SC and SCP) had the highest CEC. This may be because the compost had a higher concentration of Ca2+, K+, Mg2+ and Na+ than the biochar, and because these treatments

(SC, SCBP) do not suffer from a dilution effect as they do not contain biochar (Table 2). In turn, the base saturation in the treatments (V%) is 100%, but the aluminium saturation (Al%) is 0%, which indicates that the binding sites of the soil in these treatments are loaded with Ca2+, K+,

Mg2+ and Na+. The fact that the CEC is high and the Al% is low is important, because this indicates that it is due to the basic cations, and also that the CEC is a major controlling agent of the stability of soil structure, nutrient availability for plant growth, soil pH, and the soil’s reaction to fertilisers and other ameliorants (Hazelton and Murphy, 2007).

In depth 15-30 cm, the CEC values for times 1 and 2 behaved in the same way as in depth 0-15 cm. However, at time 3, the neutral control had the highest CEC, although if we break down this CEC it can be seen that in the final time period, despite not having the highest

CEC, the SCBP treatment did have the highest V% and lowest Al%, indicating that the exchange complex is occupied by basic cations. This demonstrates the ability of these basic cations to be retained by the biochar, something that has already been shown by Karer et al.,

(2015) and Puga et al., (2015), as biochar has a high aromaticity and high surface area, and a negative charge. In depth 30-45 cm at the first two times, the treatments made using biochar

(SCB and SCBP) had the highest CEC.

In depth 30-45 cm, SCBP had the lowest CEC, although once again if we break down the CEC by V% and Al%, we can see that SCBP had the highest V% and the lowest Al%, although in this case with only a slight difference from SCP.

4.5 Evolution of nutrients at the three depths and over the 11-month period.

Except for the case of Mg in depth 30-45 cm at times 2 and 3, in this study it was found that in general, all of the treatments increased the contents of the nutrients studied (Ca, K, Mg and Na).

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In depth 0-15 cm at times 1 and 2, the content of all of the nutrients was generally higher in the SCBP amendment, demonstrating the ability of biochar to retain these nutrients.

This can be demonstrated, as the compost has a higher content of all of the nutrients studied, except for K (Table 2); even though the compost had a higher nutrient content than the biochar, the treatment using biochar and plants was able to retain them until time 2 (7 months). At time

3, the effect of the biochar was not discernible, and was only noted for Mg. In this same time period, the SCP treatment had the highest nutrient content, possibly due to the fact that the elements used to make compost have a high level of Ca, K, Mg and Na, as has been demonstrated by authors such as Canet et al., (2008), Fernández-Hernández et al., (2014), Illera-

Vives et al., (2015) or Pérez-Esteban et al., (2012). In turn, the lower concentration of nutrients in SCBP in comparison with SCP may be because at time 3, the Brassica Juncea L. achieved their greatest biomass, leading to a higher consumption of these nutrients (Fig. 6).

In depth 15-30 cm, as the experimental period progressed, the SCBP treatment provided higher concentrations of the nutrients being studied, especially at times 2 and 3. Two exceptions were observed at time 2 in the case of K, and at time 3 in the case of Mg. This higher concentration of nutrients in the SCBP treatment at times 2 and 3 is due to the fact that although the biochar provides a smaller amount of nutrients, it has a high capacity to retain nutrients over time, retaining the nutrients provided by the compost and releasing them slowly (IBI, 2015). As a result, the benefits of applying biochar in these highly weathered systems include the prevention of nutrient loss via leaching, and the retention of nutrients in the root zone (Ippolito et al., 2012).

In depth 30-45 cm, we did not find a similar pattern to depth 0-15 cm and 15-30 cm. It should be noted that this was the deepest depth, only consisting of settling pond soil, and so it was barely affected by the addition of the treatments used. Even so, at times 1 and 2 the amendments had an effect, especially at time 2, when the SCBP treatment had the highest concentrations of the nutrients studied, except for K. The fact that SCBP had a greater effect at time 2 and not at times 1 and 3 may have been because at times 1 and 3, the biochar was not

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Anexo II able capable of significantly affecting the pH, which was lower than at time 2. SCBP had the greatest effect in retaining nutrients at time 2, when the highest pH was reached.

4.6 Limiting factors for plant production in depth 1.

In this section on the limiting factors for plant production, we will focus on height 1, as this was the height that was most affected by the treatments, and as it is the height that most affects plant development (in our case, the development of Brassica Juncea L.). The first factor we studied was Factor c, which would affect soils with a pH of less than 3.5, such as the settling pond soil. Once this soil was treated with the different amendments, it was no longer affected by Factor c. The threshold value of 3.5 was exceeded by all of the treatments throughout the entire experiment; over time, the SCB and SCBP treatments were those that maintained the highest values. Both SCB and SCBP reached an optimum pH for the development of the majority of plants; this higher pH value in the treatments using biochar was due to the high capacity of the biochar to elevate it (Hazelton and Murphy, 2007; Ohsowski et al., 2012). These optimum pH values in SCBP, compared to the lower values of SCP, may be one of the factors that led to the Brassica Juncea L. plants in SCBP having a greater amount of biomass throughout the experiment.

In the case of Factor Ca, both controls (S and SS) were affected throughout the whole the experiment, possibly due to the low Ca content in the starting materials. In this case, once the settling pond soil had been treated with the different amendments, it was no longer affected by Factor Ca. This is because the elements used to make the compost (horse and rabbit manure and seaweed) have considerable concentrations of Ca, as has been shown in a number of experiments (Canet et al., 2008, Fernández-Hernández et al., 2014, Illera-Vive et al., 2014).

According to Buol et al. (1975) and Macías and Calvo (1983), the settling pond soil would be affected by Factor e at times 1 and 2, and the neutral control at time 1. Other authors have shown that a soil CEC of less than 6 is considered very low, meaning that the soil has a low resistance to changes in soil chemistry that are caused by land use. This is one of the reasons why vegetation is unable to grow in untreated settling pond soil (Hazelton and Murphy,

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2007). As previously mentioned, if we break down the CEC in the settling pond soil, the increase in the CEC in the final time period was due to an increase of Al3+ and the fact that in this time period, its Al% was 64%.

The values for K for both controls (S and SS) are very low, and close to 0. Because of these values, both S and SS were affected by Factor k throughout the experiment. Once the different amendments were applied, the critical values for K were exceeded. Although it was observed that the concentration of this nutrient decreased over time in all of the treatments,

-1 despite this decrease the values were always above the critical value of 0.2 cmol(+)kg (Buol et al; 1975, Macías and Calvo; 1983).

As regards Factor Mg, only S and SS were affected by this factor in time 1.

The settling pond soil and neutral controls (S and SS) were seriously affected by Factor

N, as their TN contents were undetectable. None of the amendments that were applied were affected by this factor. This increase in the nitrogen content is important, as it is a vital aspect in recovering soil quality. It is also a nutrient that plays a key role in plant production, as below a certain value it becomes a limiting factor for plant production (Christensen, B.T, 2004).

4.6 Harvested biomass of Brassica juncea L.

As mentioned in the previous section, the settling pond soil (S) and the neutral control

(SS) were affected by all of the limiting factors for plant production, as none of the Brassica juncea L. planted on them grew during the experiment. Wong (2003) has already studied this obstacle to growing stable vegetation on mine soil, stating that degraded mine soils are a man- made habitat which experiences a wide range of problems for establishing and maintaining vegetation. On the contrary, in the SCP and SCBP treatments, the plants grew without any problems throughout the entire experiment. The fact that the plants grew after treating the soil has already been demonstrated by Wong (2003), who noted that the use of organic amendments is considered to be a useful tool for consolidating plant cover in settling ponds from mines.

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However, the SCPB treatment proved to be more effective than SCP, as corroborated by Fowles

(2007), who states that the use of biochar favours the positive properties of compost-type organic amendments. In the SCPB treatment, the brassicas had a larger biomass, which increased over time. The highest harvested biomass in the SCBP treatment was obtained in time

3. This was possibly due to the characteristics the biochar provided to this treatment: it is capable of reducing the leaching of nitrates, increasing the efficient use of the nutrients by the plants, increasing the pH of the soil, and improving its structure (Beesley and Marmiroli, 2011).

4.7 Principal component analysis (PCA) in the soil samples

The concentrations of the analysed nutrients at the depth 0-15 cm (Ca, Fe, K, Mg, Mn and Na) were selected to perform a principal component analysis (PCA) (Table 5) for the three time periods we studied. In the case of the principal component analysis (PCA) in the soil samples, we focused on the evolution of these components in the depth 0-15 cm over time, as it is the depth that most influences the plants that were used in this experiment. The PCA carried out for the three times explains a 95.7, 90.1 and 87.4% of the variance for the first, second and third times respectively. In all three cases, the first component is explained by a higher content of basic cations (Ca, Mg, Na and K) and a lower Fe content. In all three times, component 2 is defined by a higher Mn content.

By representing the different treatments in the components obtained for each of the times (Fig

7), it is possible to see a similar pattern for all of them. The treated soils always have positive values for component 1, while the two controls (the untreated settling pond soil and soil and sand) had negative values for this component, and therefore a higher Fe content compared to a lower basic cation content. This analysis confirms that the treatments applied to the settling pond soil significantly increased the nutrient content, especially the treatments that included

Brassica Juncea L. and biochar.

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Table 6. The component score coefficients matrix from the PCA in depth 1 Indicators PC1 PC2 Ca .973 .095

Fe -.870 -.374 K .956 -.206 Mg .974 .198 Time 1 Time Mn .007 .983 Na .971 -.201 Ca .928 .209

Fe -.878 -.299 K .950 -.119 Mg .959 .063 Time 2 Time Mn .036 .974 Na .867 -.326 Ca .996 -.015

Fe -.849 .110 K .921 .121 Mg .758 .377 Time 3 Time Mn .012 .973 Na .997 .045

Figure 7. Scatter plot with the two principal components obtained in the PCA (PC 1 and PC 2) in depth 1(0-15 cm).

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5 Conclusions

We were able to increase the pH values in the settling pond soil to which the treatments were applied, especially in the depth 0-15 cm. The treatment made using compost + biochar +

Brassica Juncea L. was the most effective in the experiment, at all of the depths.

The TC was increased in the settling pond soil after applying the treatments. We found the highest increase in the third time period, and once again the treatments with biochar were those that increased the TC the most. The TN increased considerably after applying the different treatments: in this case the most effective was the one that combined compost and Brassica

Juncea L., possibly because it did not experience a dilution effect from the biochar, and also thanks to the non-symbiont bacteria associated with the Brassica Juncea L. plants.

The CEC increased in nearly all cases after treating the settling pond soil, except in the last time in depths 15-30 cm and 30-45 cm, where it is important to note that the percentage of

Al in S was higher than in the treatments. SCBP had a higher CEC at all depths in times 1 and 2, but afterwards the effect of the biochar was gradually lost. Even so, its base saturation was always higher than in S. In general terms, the treatments applied increased the amount of nutrients in the settling pond soil. In depth 0-15 cm, the effect of the biochar was more apparent at times 1 and 2, while in depth 15-30 cm this effect was greater at times 2 and 3, possibly due to the migration of the nutrients along the cylinder. In depth 30-45 cm, the effect of the biochar was more apparent at time 2, while the amendments had the least effect on S at time 3.

Finally, the positive effect of the amendments in providing nutrients to the soil was contrasted with the principal components analysis (PCA), in which treatments SCB, SCBP and

SCP were found to be those that were most positively affected on a constant basis, especially

SCBP at times 1 and 2, and SCP at time 3; this indicates the importance of vegetation when recovering a soil of this kind. The ability of biochar to improve the conditions of a settling pond soil for planting vegetation was demonstrated, as when analysing the biomass of the Brassica

Juncea L. plants, this was greater in the treatments that combined compost and biochar. In this

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Anexo II study, we were able to conclude that biochar in has a very positive effect on enhancing the ability of compost to enhance to improve the parameters that were studied, although it may have been necessary to use a higher proportion of biochar in order for it to have had a more pronounced effect on the final depth of the cylinder over time.

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6.3. Anexo III. Using compost and technosol to decrease the bioavailable metal concentration in soil from a copper mine settling pond

Anexo III

Using compost and technosol to decrease the bioavailable metal concentration in soil from a copper mine settling pond (Environmental Science and Pollution Research, under review)

Rubén Forjána*, Alfonso Rodríguez-Vilaa, Emma F. Coveloa aDepartment of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, Lagoas, Marcosende, 36310 Vigo, Pontevedra, Spain

*Corresponding author, Tel.: +34 986812630; fax: +34 986812556. E-mail: [email protected]

Key words: Biochar, technosol, compost, phytoestabilization, settling pond soil, metal

Abstract

One of the most important sources of pollution caused by metals, if not the most important, is mining. Metal pollution is covert, persistent and irreversible. For this reason, soil metal pollution has become a severe problem in many parts of the world. The aim of this study was to observe which combination of amendments (compost+biochar or technosol+biochar) combined with Brassica Juncea L. was best at reducing the assimilable contents of Cu, and which also increased to a lesser extent the contents of other metals (Ni, Pb, Zn) found in these amendments. We also studied the phytoremediation capacity of brassicas in these amendments.

The experiment was carried out using 45 cm-deep cylinders over and 11-month period, with soil from the settling pond in the depleted copper mine located in Touro (Galicia, north-west Spain).

At depth 0-15 cm, the settling pond soil (S) had a higher CaCl2-extractable Cu, Pb, and Ni concentration, at the three time periods measured (Time 1= 3 months, Time 2= 7 months, Time

3= 11 months). The settling pond soil+technosol+biochar and vegetated with Brassica juncea L

(STBP) had the highest CaCl2-extractable concentrations of Zn over time. In general terms, the most effective treatment for reducing the phytoavailable contents of Cu, Pb, Ni and Zn was the treatment using compost+biochar + Brassica Juncea L. In the two treatments applied, Brassica

Juncea L., had a good phytostabilisation capacity.

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1. Introduction

Mineral resources are a key cornerstone for socio-economic development, meaning that the exploitation and utilization of mineral resources is essential for the modernisation of countries. Nonetheless, despite the importance of mineral resources, mineral extraction has caused serious environmental damage, especially in terms of metal pollution (Li et al., 2014).

Environmental pollution by metals has become a severe problem worldwide. Soils become increasingly contaminated by metals due to large-scale urbanization and industrialization, posing a threat to land ecosystems, surface and groundwater, as well as food safety and human health. One of the most important causes of metal pollution, if not the most important, is mining. Metal pollution is covert, persistent and irreversible (Pinto et al. 2015, Wang et al.

2001). For this reason soil metal pollution has become a severe problem in many parts of the world (Gomes et al. 2016, Solgi et al. 2012).

Because of these problems, it is important to reduce the bioassimilable content of potentially toxic elements. For years, amendments made of waste materials (compost, technosol, biochar) have been used to remediate degraded areas. Compost was one of the first amendments made of waste material that was used in the field of recovering degraded soils. They are produced by the spontaneous microbial bio-oxidation of raw wastes to produce a biologically stable, humified end product made of organic matter, using –amongst a wide range of other sources– materials such as plant and industrial agro-food wastes (Beesley et al. 2014, Bernal et al. 2007). Technosols have also been in use for several years as amendments to remediate degraded soils. These are a new group of soils that are strongly influenced by human activity.

Furthermore, they can be made from waste materials, and used in the subsequent regeneration of degraded or polluted soils. As a result, these materials are no longer considered as waste, and a value-added product is generated (Macía et al. 2014). One advantage of technosols is that they can be adjusted to the conditions of each mine waste in order to produce a specific Technosol

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Anexo III that promotes and maintains biogeochemical processes, and which reduces the availability of contaminants to plants (Santos et al. 2014). The use of biochar as an amendment is quite recent.

Its influence on soil properties also extends to the mobility of inorganic elements such as Cd,

Cu, Pb, and Zn. Due to its resistance to degradation, its effects may last for a very long time. In addition, the reduction in terms of metal bioavailability and other modifications to the substrate induced by the application of biochar may be beneficial to the establishment of a green cover on top of the wastes to acquire long-term phytostabilisation (Puga et al. 2015). Using biochar in combination with other amendments made of waste materials may enhance their positive effects

(Fellet et al. 2014, Fowles 2007).

In order to recover degraded soils such as mine soils, apart from reducing the assimilable metal content, it is important to establish a stable plant cover. Plant cover is important in improving the physical characteristics, chemical characteristics and biological characteristics of soil or wastes (Santos et al. 2014). Phytoremediation consists of using plants and associated soil microorganisms to remove or reduce contaminants in different environmental matrices, is an environmentally friendly technology that can be used to extract or immobilize metals, metalloids, and radionuclides, as well as organic xenobiotics (Pinto et al. 2015).

Phytostabilisation is a type of phytoremediation aimed at immobilising pollutants in a contaminated substrate, by establishing vegetation on top of the polluted material. This green cover prevents the pollutants being dispersed, by physically reducing wind erosion and water run-off. Through water uptake, plants can directly and indirectly lower the mobility of pollutants, thereby hindering groundwater contamination (Fellet et al. 2014). For all of these reasons, phytostabilisation is considered as an appropriate in situ green technology for the rehabilitation of mine wastes and multi-elemental contaminated soils (Santos et al. 2014).

Apart from the positive aspects we have already mentioned in this article, the re-use of organic wastes in recovering contaminated soils may also have a disadvantage, especially in the case of technosols, as some of their source materials may contribute certain amounts of metals to the soil (Yao et al. 2009). In this study we designed an experiment using cylinders to observe

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Anexo III the action of different amendments (compost+biochar, technosol+biochar) at depths from 0 to

45 cm, divided into three stages: the first from 0-15 cm, the second from 15-30 cm, and the third from 30-45 cm. The study lasted 11 months, using soil from the settling pond in a depleted copper mine located in Touro (Galicia, north-west Spain). The organic amendments selected for this study were combined with Brassica juncea L., as this plant has been used successfully for the phytoremediation of metals from polluted mine soils (Rodríguez-Vila et al. 2014).

Therefore, the aim of this study was to evaluate which combination of amendments

(compost+biochar or technosol+biochar) in conjunction with Brassica Juncea L. was best capable of reducing the assimilable contents of Cu, and which also contributes the least towards increasing the amounts of other metals contained in these amendments. We studied the phytoremediation capacity of the brassicas in combination with different amendments in a contaminated soil in order to determine whether they acted as phytoextractors, or otherwise as phytostabilisers for the metals in question.

2. Material and Methods

2.1 Soil sampling

The sample zone is located in an old copper mine in Touro, north-western Spain (8º 20' 12.06''

W 42º 52' 46.18'' N). The climate in this zone is Atlantic (oceanic) with precipitation reaching

1886 mm per year (with an average of 157 mm per month) and a mean daily temperature of

12.6ºC. The average relative humidity is 77% (AEMET, 2015). In order to carry out the study, one soil and three amendments were selected. The soil chosen came from the settling pond (S) at the Touro mine, and the three amendments were technosol (T) provided by the company

Tratamientos Ecológicos del Noroeste (T.E.N.), compost (C) provided by the company Ecocelta

Galicia S.L. (Ponteareas, Pontevedra, Spain), and biochar (B) provided by the company

PROININSO S.A.

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Ence, a cellulose production plant in Pontevedra, Spain), 10% waste from the agri-food industry

(canning plants and Ecogal), 5% sands from purification plants (sand fraction), plus a further

10% of materials whose contents are not precisely known due to the privacy policy of the company. The biochar (B) used was made from Quercus ilex wood with a pyrolysis temperature of 400 °C for 8 h.

2.2 Greenhouse experiment

The greenhouse experiment was carried out in cylinders to try to reflect the top 45 centimetres of soil. The cylinders were made of PVC, 50 cm high and with a diameter of 10 cm. A porous mesh was placed inside the cylinders, and the settling pond soil was poured onto it. The mesh used for the settling pond soil was not in contact with the PVC for any length of time. The cylinders were filled with settling pond soil (S) and the treatments:

- Settling pond soil+ Compost+biochar+ vegetated with Brassica juncea L. (SCBP).

- Settling pond soil+ Technosol+biochar+ vegetated with Brassica juncea L. (STBP).

The amendment ratios used are detailed in Table 1. The total weight of each cylinder was 3.5 kg.

Table 1. Proportions used to make the controls and the different treatments Soil Compost Technosols Biochar

S 100%

STBP 85% 11% 4%

SCBP 85% 11% 4%

The experiment was carried out over 11 months at controlled temperature and humidity

(temperature of 22±2 °C, and 65±5 % relative air humidity). A total of 45 cylinders (15 cylinders per treatment) were prepared and distributed randomly (S, SCBP, STBP). Three

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Anexo III cylinders of each type were withdrawn at 3 different times: Time 1= 3 months, Time 2= 7 months, Time 3= 11 months. The meshes were removed from the cylinders and processed for analysis at 3 different depths: the first to 0-15 cm, 15-30 cm to the second, and the third at 30-45 cm (Fig. 1). The cylinders were watered to field capacity throughout the experiment.

2.3 Soil analysis

(TN) were determined in a LECO CN-2000 module using solid samples. Exchangeable cations were extracted with 0.1 M BaCl2 (Hendershot and Duquett 1986) and their concentrations determined by ICP-OES (Optima 4300 DV; Perkin-Elmer). The phytoavailable content of copper, nickel, lead and zinc was extracted with 0.01 M CaCl2 in soil solution (Houba et al.

2000). Pseudototal metal contents were extracted with aqua regia by acid digestion in a microwave oven (Milestone ETHOS 1, Italy). Metal concentrations were determined by ICP-

AES (Optima 4300 DV; Perkin-Elmer). Pseudototal concentrations were compared with the generic reference level (GRL) established for Galician soils (Macías and Calvo de Anta 2009).

2.4 Plant growth and determination of metals in plant tissues

The Brassica juncea L. plants were pre-germinated in seedbeds until they grew two fully expanded leaves, and were then transferred to the cylinders (SCBP, STBP). The plants were harvested in the same state of maturity, for comparison in the same state of development at the three times (Time 1= 3 months, Time 2= 7 months, Time 3= 11 months). Growth was allowed under greenhouse-controlled conditions, with a photoperiod of 11:13 light/dark, a temperature of 22±2 °C and 65±5 % relative air humidity. At the end of each time period, the roots and shoots were divided and carefully washed with deionised water. Fresh biomass was weighed immediately, and dry mass was assessed after oven drying for 48 h at 80 °C and cooling at room temperature. The plant tissues, divided into roots and shoots, were air dried and

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Anexo III ground. The total concentrations of Cu, Ni, Pb and Zn in the Brassica juncea L. were extracted by acid digestion using a mixture of H2O2 and HNO3 (1:6v/v) in a microwave oven (Milestone

ETHOS 1).

In this experiment, different factors were calculated in order to discover the phytoremediation capacity of Brassica Juncea L.:

The transfer coefficient (TrC) in the plants we studied measured their efficiency to take up metals from the soil. The plant is considered to be an accumulator-hyperaccumulator biosystem when TrC > 1 (Busuioc et al. 2011). TrC was calculated using the following equation:

TrC= Cp/Cso where TC represents the transfer coefficient of the plants, Cp is the metal concentration in the shoots (mg kg-1) and Cso is the metal content of the soil (mg kg-1) (Karami et al. 2011;

Peijnenburg and Jager 2003).

The translocation factor (TF), where a high value indicates a relatively high shoot metal concentration compared to its root concentration. A plant species translocates metals effectively from the roots to shoots when TF > 1 (Baker and Brooks 1989). TF is expressed by the following equation:

TF= Cs/Cr where Cs and Cr are metal concentrations (mg kg-1) in shoots and roots, respectively.

2.5 Statistical analysis

All of the analytical determinations were performed in triplicate. The data obtained were statistically treated using version 19.0 the SPSS programme for Windows. Analysis of variance (ANOVA) and test of homogeneity of variance were carried out. In case of homogeneity, a post hoc least significant difference (LSD) test was carried out. If there was no homogeneity, Dunnett’s T3 test was performed. Student-T test was using to compare the results of TrC and TF between SCP and SCBP and the CaCl2-extractable contents of Cu, Pb, Ni, Zn between SCP and SCBP.

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3. Results

3.1 General characteristics of the settling pond soil (S), compost (C), technosol (T), and biochar (B).

The soil from the settling pond (S) had an acidic pH, while amendments compost (C), technosol (T) and biochar (B) had higher pH values (Table 2). The biochar had the highest one

(Table 2). Total carbon (TC) was significantly higher in the biochar (676±4.58 g/kg) compared to the compost (276±2.66 g/kg), technosol (256±2.51) and soil from the settling pond

(1.93±0.15 g/kg).

Table 2. Selected characteristics and metal concentrations of settling pond soil (S), compost (C), technosoil (T) and biochar (B).

S C T B GRL pH 2.73±0.08d 6.25±0.04b 6.04±0.05c 9.93±0.02a Total (g/kg) Carbon 1.93±0.15 276±2.66b 256±2.51c 676±4.58a Total (mg/kg) Nitrogen u.d 21.3±1.02a 17.6±0.50b 5.34±0.22c

- CEC (cmol(+)kg 1) 6.11±0.05d 53.5±1.07b 76.6±4.80a 15.8±0.17c Cu 637±2.08a 193±1.14c 226±5.13b 27.1±1.24d 50 Pb Pseudototal 16.1±1.00c 26.6±0.96b 89.6±1.52a u.d 80 Ni (mg/kg) 16.4±1.10c 49.7±1.71a 26.3±0.57b 25.1±2.00b 75 Zn 65.4±2.51c 403±3.33a 340±5.50b 62.6±1.70c 200 Cu 139±2.08a 0.95±0.04c 6.01±0.03b u.d Pb 0.65±0.03a 0.14±0.01c 0.33±0.02b u.d Ni CaCl2 2.25±0.30a 0.24±0.03c 1.03±0.02b 0.33±0.02c Zn (mg/kg) 64.4±1.24b 7.98±0.05c 165±1.63a 1.24±0.01d

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). u.d. undetectable level. Typical deviation is represented by ±. GRL= generic reference level (GRL) established for Galician soils (Macías and Calvo de Anta 2009).

The compost had the highest total nitrogen content (TN) and the TN was undetectable in the settling pond soil (Table 2). The exchangeable cation capacity (CEC) of C, T and B was

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Anexo III significantly higher than in the original soil. The pseudototal concentration of Cu in the settling pond soil was higher than in the used amendments (C, T and B) (Table 2). The technosol had the highest pseudototal concentration of Pb (Table 2). The compost had the highest pseudototal concentrations of Ni and Zn (Table 2). S, C and T had pseudototal Cu concentrations that were higher than the generic reference level established for Galician soils (GRL) (Macías and Calvo de Anta 2009), while T is polluted by Pb as GRL (Table 2). C and T had pseudototal Zn concentrations that were higher than the GRL for Galician soils (Table 2). The extractable

CaCl2 concentration of Cu, Pb and Ni in S was significantly higher than in C, T and B (Table 2).

The CaCl2-extractable concentration of Zn in T (165±1.63 mg/kg) was higher than in S

(64.4±1.24 mg/kg), C (7.98±0.05) and B (1.24±0.01). The biochar had no detectable CaCl2- extractable concentrations of Cu and Pb (Table 2).

3.2 Evolution of the pseudototal concentrations of Cu, Pb, Ni, and Zn at three depths and over the 11-month period.

In general, the settling pond soil (S) had the highest Cu pseudototal concentration over time and at different depths, except in time 3 at the 30-45 cm depth (Fig. 1A, 1B, 1C, 1D, 1E,

1F).

In the 0-15 cm depth, the pseudototal concentrations of Pb, Ni and Zn were higher in

STBP than in S and SCBP (P < 0.05) (Fig. 1A, 1B). In the 15-30 cm depth, SCBP had the highest pseudototal concentrations of Pb over the time (Fig 1C, 1D). The pseudototal concentration of Ni was higher in treatment SCBP at time 1 than in S and STBP, but at time 3

STBP had the highest peseudototal concentration of Ni (P < 0.05) (Fig. 1C, 1D). S had the highest pseudototal concentration of Zn over the time in this depth (Fig. 1C, 1D). In the 30-45 cm depth, S had the highest pseudototal concentrations of Pb over the time (Fig 1E, 1F). The pseudototal concentration of Ni was higher in treatment SCBP at time 1 than in S and STBP,

STBP had the highest pseudototal concentration of Ni at time 3 (P < 0.05) (Fig. 1E, 1F). S and

STBP had the highest pseudototal concentration of Zn at time 3 respectively (P < 0.05) (Fig. 1E,

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1F).

3.3 Evolution of CaCl2-extractable (Phytoavailable) contents of Cu, Pb, Ni, and Zn at three depths and over the 11-month period.

At depth 0-15 cm, the settling pond soil (S) had a higher CaCl2-extractable Cu, Pb, and

Ni concentration, at the three times (P < 0.05) (Table 3). STBP had the highest CaCl2- extractable concentrations of Zn over the time (Table 3).

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Table 3. Evolution of CaCl2-extractable (Phytoavailable) concentrations Cu, Pb, Ni, Zn (mg.kg-1) at depth 0-15 cm and over the 11-month period.

Depth 0-15 cm S STBP SCBP

Cu 50.6±3.25a 2.54±0.10b 0.74±0.02c Pb 0.41±0.07a 0.30±0.02b 0.13±0.01c Ni 2.06±0.05a 1.54±0.16b 1.27±0.37b Time 1 Zn 3.09±0.87c 145±3.54a 4.72±0.42b

Cu 54.5±6.82a 1.77±0.06b 0.58±0.89c Pb 0.44±0.02a 0.21±0.01b 0.11±0.03c Ni 2.53±0.041a 1.55±0.29b 0.67±0.12c

Time 2 Zn 2.63±0.09c 134±3.69a 6.05±0.89b

Cu 54.1±3.13a 1.22±1.00b 0.46±0.01c Pb 0.44±0.04a 0.35±0.02b 0.15c Ni 3.32±0.25a 0.93±0.01b 0.04±0.02c Time 3 Zn 2.34±0.70c 151±4.02a 6.92±0.47b

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). Typical deviation is represented by ±

At depth 15-30 cm, S had the highest CaCl2-extractable concentration of Cu over the time (Table 4). STBP had the highest CaCl2-extractable concentration of Zn over the time (P <

0.05) (Table 4). At time 1, S and STBP had higher CaCl2-extractable concentration of Pb than in

SCBP; STBP had the highest CaCl2-extractable concentration of Ni (Table 4). At time 2, S had the highest CaCl2-extractable concentrations of Pb, and SCBP had the highest CaCl2-extractable concentrations of Ni (P < 0.05) (Table 4). At time 3, STBP and SCBP had the highest CaCl2- extractable concentrations of Pb and Ni respectively (Table 4).

Table 4. Evolution of CaCl2-extractable (Phytoavailable) concentrations Cu, Pb, Ni, Zn (mg.kg-1) at depth 15-30 cm and over the 11-month period.

Depth 15-30 cm S STBP SCBP

Cu 50.9±1.31a 41.9±1.00b 40.8±0.01c Pb 0.40±a 0.41±0.02a 0.32±0.03b Ni 2.06±0.07c 3.21±0.17a 2.95±0.03b Time 1 Zn 2.37±0.06c 4.50±0.06a 3.20±0.04b

Cu 55.4±0.33a 44.0±1.68c 46.3b Pb 0.48±0.01a 0.44±0.02b 0.31±0.02c Ni 2.40±0.01c 2.88±0.07b 3.44±0.13a Time 2 Zn 2.59±0.01b 3.23±0.20a 2.29±0.04c

Cu 55.9±0.03a 48.1±1.01b 48.1±0.06b Pb 0.43±0.01b 0.50±0.01a 0.39c Ni 3.31±0.12b 2.74±0.05c 3.68±0.07a Time 3 Zn 2.58±0.17c 3.62±a 2.95±0.06b

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). Typical deviation is represented by ±.

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At depth 30-45 cm, at time 1 SCBP had the highest CaCl2-extractable concentrations of

Cu and Pb. S and SCBP had the highest CaCl2-extractable concentrations of Pb and Ni respectively (P < 0.05) (Table 5). At time 2, the settling pond soil (S) had the highest CaCl2- extractable concentrations of Cu and Pb. SCBP had higher highest CaCl2-extractable concentration of Ni. The CaCl2-extractable concentration of Zn was higher in S and STBP than in SCBP (P < 0.05) (Table 5). At time 3 S had higher CaCl2-extractable concentrations of Cu,

Pb, Ni and Zn than in STBP and SCBP (P < 0.05) (Table 5).

Table 5. Evolution of CaCl2-extractable (Phytoavailable) concentrations Cu, Pb, Ni, Zn (mg.kg-1) at depth 30-45 cm and over the 11-month period. Depth 30-45 cm S STBP SCBP

Cu 52.0±0.01b 50.1±0.01c 54.3±0.05a Pb 0.55±0.02a 0.32±0.03b 0.33b Ni 2.16±0.05c 3.01±0.02b 3.75±0.01a Time 1 Zn 3.45±0.02b 3.72±0.01a 2.10c

Cu 53.1±1.76a 50.2±1.64c 52.9±0.02b Pb 0.55±0.49a 0.37±0.29ab 0.33b Ni 2.57±1.02b 2.75±0.79b 3.59±0.05a Time 2 Zn 3.43±0.03a 3.55±0.50a 2.00±0.18b

Cu 58.3±1.11a 46.3±0.83b 47.4±0.04b Pb 0.47±0.01a 0.43±0.02b 0.31±0.03c Ni 3.48±0.09a 2.67±0.10c 3.03±0.05b Time 3 Zn 3.73±0.24a 3.06±0.02b 2.48±0.01c

For each row, different letters in different samples means significant differences (n=3, ANOVA; P<0.05). Typical deviation is represented by ±

3.4 Harvestable amounts of Cu, Pb, Ni, Zn and determination of metals in plant tissues: translocation factor (TF) and transfer coefficient (TrC).

Harvestable amounts of Cu, Pb, Ni, and Zn in Brassica juncea

The Brassica juncea L. was not capable of growing in the settling pond soil (S), and for this reason is not shown in figure 2. On the one hand, the concentrations of Pb and Zn in the roots was higher in STBP than in SCBP, on the other hand the concentrations of Cu and Ni showed no significant difference between treatments (Fig. 2B; p < 0.05). In the second harvest

(Time 2), the concentrations of Cu in the Brassica juncea L. shoots showed no significant difference between STBP and SCBP. STBP had higher values of Pb and Zn concentrations in

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Anexo III shoots than SCBP. The concentration of Ni in the shoot was higher in SCBP than in STBP (Fig.

2C; p < 0.05). The concentrations of Cu, Pb and Zn in the roots were higher in STBP than in

SCBP (Fig. 2D; p < 0.05). In the final harvest (Time 3), STBP had the highest concentrations for Pb, Ni and Zn in shoots, but the concentration of Ni showed no significant difference between treatments (Fig. 2E; p < 0.05). As in the first harvest, the concentrations of Pb and Zn in the roots was higher in STBP than in SCBP, and the concentrations of Cu and Ni showed no significant difference between treatments (Fig. 2F; p < 0.05).

Fig. 2 Harvestable amounts of Cu, Pb, Ni, Zn and determination of metals in plant tissues. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represent standard deviation

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Translocation factor (TF)

The TF at time 1 for Cu, Ni, and Zn was the highest in the settling pond soil that was treated with compost and biochar (SCBP); the TF for Pb was higher in the settling pond soil that was treated with technosol and biochar (STBP) (Fig. 3A). At time 2, the TF for all metals except Zn was the highest in STBP (Fig. 5B). (P < 0.05) (Fig. 3B). The TF at time 3 for Cu did not reveal any differences between the different treatments, although the TF for Pb Ni and Zn was significantly higher in STBP than in SCBP (Fig. 3C).

Fig. 3 Translocation factor (TF) of Cu, Ni, Pb, and Zn to and within mustards according Translocation factor (TF) (shoots concentration/roots concentration) over the 11- month period. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represent standard deviation.

Transfer coefficient (TrC)

At time 1, the TrC for Cu, Pb Ni and Zn was significantly higher in SCBP than in STBP

(Fig. 4A). At times 2 and 3, STBP had the highest TrC for Pb and Zn. SCBP had the highest values of TC for Cu and Ni. The TC for Cu did not reveal any significant differences between the different treatments (P < 0.05) (Fig. 4B, 4C).

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Fig. 4 Transfer of Cu, Ni, Pb and Zn to and within mustards according to transfer coefficient (TrC) (shoots concentration/pseudo-total soil concentration) over the 11-month period. For each row, differ letters in different samples means significant differences (n=3, Student’s t test: P< 0.05). Error bars represent standar deviation.

4. Discussion

4.1 Evolution of the pseudototal contents of Cu, Pb, Ni, Zn at three depths and over the

11-month period.

(STBP) than in the treatment with compost (SCBP). This is possibly due to the materials used to make the technosol, such as sewage sludge or waste from the agri-food industry plants

(Alvarenga et al. 2016, González-González et al. 2013). It should be noted that although the compost had more pseudototal Ni and Zn than the technosol (Table 2), these increased more in the STBP treatment than in the SCBP treatment, as previously mentioned. At this stage, the pseudototal Zn contents increased the most, due to the elements used to make the technosol and compost. These waste materials, such as the purification plant sludge, usually have high pseudototal Zn concentrations (Pérez-Esteban et al. 2014, Rorat et al. 2016, Tai et al. 2016).

Despite the fact that at the end of time 3 the pseudototal contents of Pb, Ni and Zn increased

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Anexo III after applying the treatments, it is important to note that the percentages of phytoavailable concentrations with respect to the pseudototal were higher in S than in STBP or SCBP. An exception to this is the Zn in the STBP treatment; also, these percentages were higher in STBP than in SCBP. The percentages of phytoavailable concentrations with respect to the pseudototal for S were 1.5% Pb, 32.5% Ni, 6.8% Zn, while the percentages for SCBP were 0.4% Pb, 0.4%

Ni, 3.1% Zn. In the case of STBP the percentages were 0.4% Pb, 2.5% Ni, 31% Zn. This shows that these increases in the pseudototal contents caused by the treatments are relative, and not so problematic.

At depth 15-30 cm at the end of time 3, there was an increase in the pseudototal contents of Ni and Pb in both treatments (STBP and SCBP). This is to be expected, because as may be seen in

Table 2, both the compost and technosol have more pseudototal contents of these elements. If we compare the percentages of the phytoavailable concentrations with respect to the pseudototal for Ni and Pb, we can see that these percentages were once again higher in S than in STBP and

SCBP. In this case, the percentages of phytoavailable concentrations with respect to the pseudototal for S were 1.2% Pb, 29% Ni, while the percentages for SCBP were 1.1% Pb, 24%

Ni. In the case of STBP, the percentages were 1.5% Pb, and 15% Ni.

At depth 30-45 cm, the pseudototal content of Pb, Ni and Zn increased more with the STBP treatment than with the SCBP and S treatments, except in the case of Pb, whose pseudototal content did not vary between S and STBP. As occurred at depth 0-15 cm and 15-30 cm, the percentages of phytoavailable concentrations with respect to the pseudototal content were higher in S than in the treatments. In this case, the percentages of phytoavailable concentrations with respect to the pseudototal for S were 1.3% Pb, 28% Ni, and 9.5% Zn, while the percentages for SCBP were 0.9% Pb, 19% Ni, and 5.8% Zn. In the case of STBP, the percentages were 1.2% Pb, 13% Ni, and 6.8% Zn. At this depth it is important to consider that it was equally affected by the metals in the settling pond soil, as at a depth of 30-45 cm the contents were entirely settling pond soil, and by the contents that migrated from the 0-15 cm depth, containing the majority of the amendments.

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This reduction in the percentage of phytoavailable concentrations with respect to the pseudototal of Cu, Pb and Ni, Zn is due to the capacity of the compost, technosol and biochar to fix these metals, and is also possibly due to the contribution of organic matter and an increased pH caused by the treatments. Some authors like Cui et al. (2016) or Gusiatin et al. (2016) found relationships between the activity or solubility of several metals and the soil properties of pH and organic matter content.

4.2 Evolution of the CaCl2-extractable (Phytoavailable) concentrations of Cu, Pb, Ni,

Zn at three depths and over the 11-month period

The effect of the different treatments is clearer at depth 0-15 cm, especially in the case of the treatments SCBP, which generally have the lowest values for phytoavailable concentrations of Cu, Pb, Ni and Zn. In turn, the STBP treatment reduced the phytoavailable concentrations of Cu, Pb and Ni, but not of Zn. This decrease in the assimilability of the potentially toxic elements with the SCBP treatment is due to the fact that the compost had a significantly higher pH, and a higher TC concentration than the technosol (P<0.05), which is enhanced by the effect of the biochar. Other authors such as Fowles (2007) have previously observed a greater positive effect on the soil properties by applying organic amendments that contained biochar amongst their components, compared with organic amendments that did not contain biochar. The positive effect of the compost and technosol is due to the residues used to make it, such as waste from agri-food industries, seaweed in the case of compost or sludge from a wastewater treatment plant, ash, waste from the agri-food industry and sands from purification plants in the case of technosol. Several authors have demonstrated that these wastes have a high organic matter content and high concentrations of basic cations. A direct effect of this high basic cation content is a high CEC and pH (Ohsowski et al. 2012, Pérez-Esteban et al. 2012). It should be noted that compost contains a special component –seaweed– which influences reducing the phytoavailable content of trace elements. This is because biosorption in algae has mainly been attributed to the cell wall, composed of a fibrillar skeleton and an amorphous embedding matrix. Both electrostatic attraction and the complexation of metals in the

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Anexo III biomaterial can play an important role in reducing the bioavailable concentrations of metals

(Figueira et al. 2000). As previously mentioned, the positive characteristics of the compost and technosol were improved by the biochar, as it has a negative charge, and so the cation exchange capacity (CEC) may increase due to the induced higher pH, and in turn, the electrostatic attraction. Biochar also has a large surface area and a high organic matter content (Karer et al.

2015, Puga et al. 2015, Beesley and Marmiroli 2011).

At the 15-30 cm depth, the effect of the treatments was not so clear. In fact, at time 1, the settling pond soil to which the treatments were applied generally had a higher phytoavailable concentration of Ni and Zn. However, on comparing between STBP and SCBP, the latter once again had the lowest values for the phytoavailable concentrations of Pb, Ni and

Zn (P < 0.05). This positive effect in SCBP is possibly due to the increased pH and organic matter content (the compost and biochar had very high TC and pH values; Table 2), which play a crucial role in metal solubility, causing the metals not to be in a phytoavailable form in the soil

(Cui et al. 2016, Gusiatin et al. 2016, Karer et al. 2015). At time 2 and 3, the SCBP treatment was generally able to more effectively reduce the phytoavailable concentrations of Cu, Pb, and

Zn, but not that of Ni (Table 4). This failure to reduce the phytoavailable concentration of Ni is possibly due to the fact that the base material used for the compost has much higher pseudototal concentrations of this element than S. This means that by not being able to sufficiently increase the pH and TC content at the depth of 15-30 cm, then the phytoavailable concentrations increase. By observing the situation at the depth of 15-30 cm over time, it was found that the effect of the compost+biochar+Brassica Juncea L. in reducing the phytoavailable concentrations was greater than that of the technosol+biochar+Brassica Juncea L.

At the 30-45 cm depth in time 1 and 2, the effect of the treatments was not clear, especially in time 1. This is possibly because in time 1, the treatments at this depth had still not had any effect, as the amendments were deposited on the surface, and it takes time for the organic matter and basic cations to migrate that help to increase the pH value. In time 2, it can be seen that the treatments applied began to take effect on the phytoavailable concentrations of

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Anexo III the potentially toxic elements, which were generally higher in S. By time 3, the effect of the amendments was very clear, with both having lower phytoavailable concentrations than S. This is because C, T and B all have a much higher TC, pH and CEC than the settling pond soil (see

Table 2). Over time, as the cylinders were watered and the treatments settled, this led to part of the amendments migrating through the profile and causing an increase in the TC, pH and CEC.

These increases have a positive effect on the reduction of the phytoavailable concentrations, as has been demonstrated by various authors (Cui et al. 2016, Gusiatin et al. 2016, Karer et al.

2015).

4.3 Uptake and transfer of metals to Brassica Juncea L. plants

Harvestable amounts of Cu, Pb, Ni, and Zn.

At time 1, the harvestable amounts of Cu, Ni and Zn in the shoots were higher in the brassicas harvested in SCBP, and the harvestable amounts of Pb in the shoots was higher in those harvested in STBP. This may be due to the short amount of time that had transpired since the treatment was applied. If we consider the situation over time, this behaviour was reversed, and at time 3 the harvestable amounts of Pb, Ni and Zn in the shoots was higher in the brassicas harvested from the soil treated with technosol and biochar. The fact that the harvestable amounts of the metals studied were lower in SCBP may be due to the fact that the compost has a higher TC content, higher pH, and lower phytoavailable concentrations of the metals being studied (Table 2). Violante et al. (2010) already concluded that factors such as an increased pH and the presence of organic ligands have a positive effect on phytoavailability. Out of the four metals studied, the highest harvestable amounts in the shoots were of Zn; this is because both amendments (compost and technosols) have very high Zn concentrations (Table 2), and also because Zn is a metal with less affinity for organic matter in comparison with others such as Cu or Pb (Forján et al. 2016).

In general, the harvestable amounts of Cu, Pb and Zn in the roots, with the exception of

Ni, were higher in the brassicas harvested in STBP. This shows that the phytoavailable content

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Anexo III is higher in this treatment. If we once again consider tables 3 and 4, we can see that at the depths of 0-15 cm and 15-30 cm, the STBP treatment had higher concentrations of the metals studied than STBP. Therefore, the effect of the biochar is not the same in combination with both amendments. At the end of the experimental period we were able to observe the effect of the biochar, as time 3, when the treatments were most settled, was when the harvestable amounts in the roots were the highest. This has already been described by authors such as Prapagdee et al.

(2014) who demonstrated that the application of biochar in metal-contaminated soil for improving plant growth can also lead to an increase in metal uptake by some plants, and in our case this effect was greater in the combination using compost+biochar than in the combination of technosol+biochar.

Transfer coefficient (TrC)

According to Busuioc et al. (2011), a plant is considered as having a phytoextraction capacity when the TrC value is greater than 1. In our case, the brassicas that grew on SCBP only had a value close to 1 in the case of Zn in time 1. Therefore, in the case of the four metals we studied (Cu, Pb, Ni, Zn), the plants that grew on SCBP and STBP did not have any phytoextracting capacity. However, apart from indicating that the brassicas are not phytoextractors in these treatments, these low TrC values also reveal something quite important, which is that they have a good phytostabilising capacity. The low TrC values indicate that brassicas could act as a phytostabilising plant for Cu, Pb, Ni and Zn, as phytostabilising plants are charcacterised by having a low root to shoot transfer coefficient (Kidd et al. 2009). This phytostabilisation capacity of the brassicas in the mine settling pond soil is important, as a typical scenario in which phytostabilisation can be considered is represented by metalliferous sites (abandoned mining sites), where the presence of wastes and mine tailings results in severe pollution and causes a major visual impact on the local environment.

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Translocation factor (TF)

Throughout the experiment, the TF values followed a pattern whereby as the time progressed, the TF values in STBP increased. However, it is true that these values only exceeded the value of 1 for Pb and Ni in time 2, a value which according to Baker and Brooks

(1989) means that the plant is capable of translocating metals effectively from the roots to the shoots. The fact that the TF values are less than one with the exception of these two cases, in the same way as the TrC, provides us with a great deal of information. These low TF values confirm that in this case, Brassica juncea L. behaves as a phytostabiliser, as could be deduced from the TrC values. As noted by Pinto et al. (2015) an important feature for a plant to be a phytostabiliser is that they should be poor translocators of metals to aboveground tissues. This was also demonstrated by Nouri et al. (2009), who noted that phytoremediating plants with low

TF values fix metals in the root, thereby acting as phytostabilisers.

In our study, based on the data for TrC and TF, apart from two isolated exceptions, we observed that in the two treatments applied to a mine soil, Brassica Juncea L. has a good phytostabilisation capacity in both treatments, as was also deduced from the TrC data. This phytostabilisation capacity is combined with a phytoextraction capacity for Pb and Zn in time 2 in the brassicas from the STBP treatment, possibly because STBP had higher phytoavailable concentrations of Pb and Zn than SCBP.

5 Conclusions

At depth 0-15 cm, the STBP and SCBP amendments reduced the phytoavailable contents of Cu, Pb and Ni. SCBP was the most effective treatment, providing lower phytoavailable contents of Cu, Pb, Ni and Zn in comparison to STBP. At depth 15-30 cm, the effect of the treatments was not so clear, although the SCBP treatment was generally more effective at reducing the phytoavailable concentrations of Cu, Pb, and Zn. At depth 30-45 cm, it was found at the end of the 11-month period that both treatments were effective in terms of reducing the phytoavailable contents of Cu, Pb, Ni and Zn. In general terms, the treatment

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Anexo III combining compost+biochar+Brassica juncea L. was the most effective at reducing the phytoavailable contents of Cu, Pb, Ni and Zn. According to the data obtained for TrC and FT, we can conclude that in general, Brassica juncea L. has a good phytostabilisation capacity when grown on these treatments. Also, two isolated exceptions were observed, where this phytostabilisation capacity was combined with a phytoextraction capacity for Pb and Zn in time

2, in the brassicas in the STBP treatment.

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6.4. Anexo VI. Comparative effect of compost and technosol enhanced with biochar on the fertility of a mine soil

Anexo IV

Comparative effect of compost and technosol enhanced with biochar on the fertility of a mine soil (Pedosphere, under review)

Rubén Forjána, Alfonso Rodríguez-Vilaa, Emma F. Coveloa, Verónica Asensiob* a Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, As Lagoas-Marcosende,

36310 Vigo, Pontevedra, Spain. b* Corresponding author: Department of Plant Nutrition, University of São Paulo – Center of Nuclear Energy in

Agriculture (USP-CENA), Av. Centenário 303, 13400-970, Piracicaba, SP, Brazil.

*Corresponding author, Tel.: +34 986812630; fax: +34 986812556. E-mail: [email protected]

Abstract

A large number of studies on the reclamation of mine soils focused on the problem caused by metals, and did not explore in depth the issue of nutrients and vegetation after the application of organic materials. The aim of this study was to compare the effect of two treatments made of wastes and vegetated with Brassica juncea L. on the fertility of a settling pond mine soil. The first treatment was compost, biochar and B. juncea (SCBP), and the second treatment was technosol, biochar and B. juncea (STBP). This study evaluated the effect of the treatments on the soil nutrient concentrations and fertility conditions in the soil-amendment mixtures, after 11 months of greenhouse experiment. Total carbon and nitrogen concentrations were higher in treatment SCBP than in treatment STBP after 7 months but, after 11 months, carbon concentration was higher in STBP. The used technosol could have forms of carbon more stable than compost, which could be released slower than in the compost-amended soils. Both compost and technosol mixed with biochar also increased the concentration of calcium, potassium, magnesium and sodium in exchangeable form in the mine soil.

Keywords: settling pond soil, compost, technosol, biochar, soil reclamation, soil nutrients.

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1. Introduction

Metal mining areas are an environmental concern due to the high levels of potentially toxic elements, such as metals, that can be transported to surrounding environments through wind erosion, leaching to surface water or to groundwater (Puga et al., 2016a). Phytoremediation has been proposed as a suitable strategy to decrease the environmental risks of metal polluted mine soils (Parraga-Aguado et al., 2015). Phytostabilization provides a long-term vegetal cover that may mitigate erosion and considers the use of metal tolerant plant species to immobilize metals

(Conesa et al., 2006). However, mine soils usually show several characteristics that may limit plant growth, including extreme pH values, low fertility conditions, limited water holding capacity and high metal concentrations, among others (Parraga-Aguado et al., 2015). Therefore, metal pollution on mine soils should be effectively controlled and ameliorated.

There is a general assumption that organic amendments may enhance soil fertility, structure and plant cover. The performance of organic amendments depends on their nature, the dose and the properties of the polluted soil (Parraga-Aguado et al., 2015). Organic amendments, such as technosol made of different wastes, compost and biochar, have been used for soil remediation purposes due to their ability to reduce the availability of potentially toxic elements (Puga et al.,

2015b; Rodríguez-Vila et al., 2016; Venegas et al., 2016). Thus, the use of these organic materials as soil amendment may be a highly practical and low cost strategy to remediate polluted soils through changing the mobility and availability of metals.

Organic amendments made of wastes and used in soil reclamation can be classified as technosols according to the (IUSS, 2014). Technosols are dominated or strongly influenced by human made materials and contain a significant amount of artefacts (IUSS Working Group

WRB, 2014). Biochar is an organic carbon-rich material produced from the pyrolysis of biomass under an oxygen-limited environment (Lehmann and Schad, 2007). Biochar source materials are generally limited to biological residues (e.g. wood, poultry litter and crop residues) and not commonly activated or further treated before application to soils (Beesley et al., 2011).

The use of biochar for soil remediation is recommended due to its low cost and its relative

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Anexo IV stability in the environment, which could render metal immobilization for longer periods as compared to other organic materials (Beesley et al., 2011).

Compost is a complex matrix rich in organic matter, especially in humic acids, which is considered as a great alternative for waste management (Gandolfi et al., 2010). Composting refers to the process of humification and stabilization of organic wastes (e.g., sewage sludge, manure, municipal solid waste and green waste) (Huang et al., 2016). The addition of compost to polluted soils can reduce metal mobility and restore soil biological and physical properties

(Manzano et al., 2016). The organic matter of compost is expected to decrease the metal availability in soil due its high content of functional groups, capable of forming strong complexes with metal cations (Manzano et al., 2016). With compost application, both the physical structure and fertility of soil are improved, microbial activity is enhanced and plant biomass is increased (Huang et al., 2016). Mineral ions, humic substances, and microorganisms in compost considerably influence the immobilization of metals and the reduction of the ecological and environmental risk of these elements in polluted soils (Huang et al., 2016).

Previous studies about remediation of mine soils with organic amendments have focused on reduce the concentration of metals, but only some of them evaluated the nutrient concentrations of the mine soil after the application of organic materials. The purpose of this study was to evaluate the effect of two treatments made with different mixtures of compost, technosol and biochar and vegetated with Brassica juncea L. on the soil nutrient concentrations and fertility conditions in a settling pond mine soil during 11 months of greenhouse experiment.

2. Material and Methods

2.1. Experimental site, soil sampling and amendments

The sample zone is located in an old copper mine at Touro, northwestern Spain (8º 20' 12.06'' W

42º 52' 46.18'' N) (Fig. 1). The climate in this zone is Atlantic (oceanic) with precipitation reaching 1886 mm per year (with an average of 157 mm per month), a mean daily temperature of 12.6ºC and an average of relative humidity of 77% (AEMET, 2016). In order to carry out the study, one soil and three amendments were selected. The soil belonged to a settling pond (S) at

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Anexo IV the Touro mine. The three amendments were technosol (T) provided by the company

Tratamientos Ecológicos del Noroeste (T.E.N.), compost (C) provided by the company Ecocelta

Galicia S.L. (Ponteareas, Spain) and the biochar (B) provided by the company PROININSO

S.A.

Fig. 1 Location of the sampled area in Touro mine.

The settling pond soil (S) was made of waste material resulting from the flotation of sulphides during copper processing. The pool has been dry for several years, and is considered a

Technosol according to the latest update of the World Reference Base for Soil Resources (IUSS

Working Group WRB, 2014). The compost (C) consisted of horse and rabbit manure mixed with grass cuttings, fruit and seaweed. The technosol (T) was a mixture of 60% sewage sludge from a wastewater treatment plant, 10% sludge from an aluminum factory (Padrón, Spain), 5% ashes from a cellulose factory (ENCE company, Spain), 10% residues from agri-food companies, and 5% sand from a wastewater treatment plant. It is missing 10% from 100% due to the company's privacy policy. The biochar (B) used was made from Quercus ilex wood with a pyrolysis temperature of 400°C for 8h.

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2.2. Greenhouse experiment

The greenhouse experiment was carried out in cylinders to try to reflect the top 45 cm of soil.

The cylinders were made of PVC with a height of 50 cm and a diameter of 10 cm. A porous mesh was introduced into the cylinders, and the settling pond soil into the inner. The mesh was used for the settling pond soil was not in contact with the PVC for a long period of time (Fig. 2).

Fig. 2. Cylinder design and the different depths collected

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The cylinders were filled with settling pond soil (S, control sample) and the treatments:

- Settling pond soil (S)

- Settling pond soil + Compost + Biochar + Brassica juncea L. (SCBP).

- Settling pond soil + Technosol + Biochar + Brassica juncea L. (STBP).

The used amendment ratios are detailed in Table 1. The final total weight of each cylinder was

3.5 kg.

Table 1. Proportions used to make the controls and the different treatments.

Soil Compost/Technosols Biochar

S 100%

SCBP 85% 11% 4%

STBP 85% 11% 4%

The experiment was carried out for 11 months at controlled temperature and humidity

(temperature of 22 ± 2°C and 65 ± 5% relative air humidity). A total of 45 cylinders (15 cylinders for each treatment) were prepared and randomly distributed (Fig. 2). Three cylinders of each type were withdrawn at three different times: time 1 = 3 months, time 2 = 7 months and time 3 = 11 months. The meshes were removed from the cylinders and processed for analysis by separating three different depths: 0-15 cm the first, 15-30 cm the second and 30-45 cm the third

(Fig. 2). The cylinders were maintained to field capacity throughout the whole time of the experiment.

2.3. Soil analyses

Soil samples collected from the cylinders were air dried, passed through a 2 mm sieve and homogenized prior to analysis. Soil pH was determined using a pH electrode in 1:2.5 water to soil extracts (Porta, 1986). Total soil carbon (TC) and total nitrogen (TN) were determined in a

LECO CN-2000 module using solid samples. Exchangeable cations (Ca2+, K+, Mg2+, Na+ and

3+ Al ) were extracted with 0.1 M BaCl2 (Hendershot and Duquette, 1986) and element concentrations were determined by ICP-OES (Perkin-Elmer Optima 4300 DV). Effective cation

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Anexo IV exchange capacity (CEC) was calculated with the sum of exchangeable cation concentrations.

Pseudototal metal contents were extracted by acid digestion with aqua regia in a microwave oven (Milestone ETHOS 1, Italy). Metal concentrations were determined by ICP-OES.

2.4. Plant growth

The Brassica juncea L. were pre-germinated in seedbeds until they grew two fully expanded leaves, and then transferred to the cylinders with the settling pond mine soil (S) and treatments

(SCBP and STBP). They were not planted in the soil without amendment because previous studies shown that they are not able to grow in this type of soil. The plants were harvested in the same state of maturity, for comparison in the same state of development in the three times

(Time 1 = 3 months, Time 2 = 7 months, Time 3 = 11 months). Growth was allowed under greenhouse-controlled conditions, with a photoperiod of 11:13 light/dark. At the end of each time, the height of the plants was measured and then they were carefully washed with deionized water. Fresh biomass was immediately weighed, and dry mass was assessed after oven-drying for 48h at 80°C and then cooling at room temperature. The plant tissues, divided into roots and shoots, were air-dried and ground for analyses.

2.5. Statistical analysis

All analytical determinations were performed in triplicate. The data obtained were statistically treated using the program SPSS version 19.0 for Windows. Analysis of variance (ANOVA) and test of homogeneity of variance were carried out. In case of homogeneity, a post hoc least significant difference (LSD) test was carried out. If there was no homogeneity, Dunnett’s T3 test was performed. Principal component analyses (PCA) were also carried out.

3. Results

3.1. General characteristics of settling pond soil (S), compost (C), technosol (T) and biochar (B)

Biochar had higher pH and total carbon concentrations than the settling pond soil (S), technosol

(T) and compost (C) (P < 0.05) (Table 2). The soil even had an acidic pH. The compost had the

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Anexo IV highest total nitrogen content (TN) (P < 0.05) (Table 2). The TN values were undetectable in S

(Table 2). The technosol had the highest Ca, Mg and Na concentrations, and the biochar had the highest concentrations of K (Table 2). The cation exchange capacity was significantly higher in

T than in the settling pond soil, compost and biochar (P < 0.05) (Table 2). The soil had the highest pseudototal concentration of Cu. The technosol had the highest pseudototal concentrations of Ni and Zn. Both C and S had the highest pseudototal concentrations of Pb

(Table 2).

Table 2. Characteristics of the mine tailing (S), technosoil (T), compost (C) and biochar (B).

S T C B

pH 2.73±0.07d 6.04±0.05c 6.47±0.02b 9.90±0.02a

TC g kg-1 1.93±0.15d 256±2.51c 276±2,49b 676±4.58a

TN mg kg-1 u.l. 17.6±0.50b 21.3±1.02a 5.34±0.22c

Ca 13.3±0.02d 7785±0.15a 6455±153b 531±4.88c

K 6.40±0.89d 2687±0.08c 3041±46.5b 3243±23.1a mg kg-1 Mg 216±2.10d 1997±0.25a 1038±14.9b 548±11.6c

Na 27.4±0.90d 2805±0.03a 987±12.4b 65.4±0.07c

-1 CEC cmol(+)kg 6.11±0.05d 76.6±0.04a 53.5±1.07b 15.8±0.17c

Cu 637±2.08a 193±1.14c 226±5.13b 27.1±1.24d

Pb Pseudototal (mg 16.1±1.00c 26.6±0.96b 89.6±1.52a u.l.

-1 Ni kg ) 16.4±1.01c 49.7±1.71a 26.3±0.57b 25.0±2.00b

Zn 65.4±2.51c 403±3.33a 340±0.50b 62.6±1.95d

Means±SD. For each row, different letters in different samples means significant differences (n=3, P < 0.05). u.l.: undetectable level.

3.2. Evolution of the pH and the cation exchange capacity (CEC)

At 0-15 cm depth, during the 11 months of the experiment, SCBP and STBP had significantly higher pH values than S. STBP had the highest values of pH at time 1-2, but at time 3 there are no significant differences between them (Table 3). At 15-30 cm depth, at time 1 the soil had significantly higher pH than SCBP and STBP (P < 0.05). At time 2, the STBP had the highest pH and at time 3, SCBP had the highest. At 30-45 cm depth, at time 1-2, the soil had the significantly highest pH values but, at time 3, SCBP had the highest value (P < 0.05).

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At 0-15 cm depth, the cation exchange capacity (CEC) was always significantly higher in the amended soils than in S (Table 3). At time 1 and 2, SCBP had the highest CEC but, at time 3,

SCBP and STBP did not show significant differences (P < 0.05). At 15-30 and 30-45 cm, at times 1 and 2, SCBP had the highest CEC. In the time 3, the soil had the highest CEC.

-1 Table 3. Evolution of pH and cation exchange capacity (CEC) (cmol(+)kg ) in three depths and at 3, 7 and 11 months of experiment (Time 1, 2 and 3, respectively).

pH S SCBP STBP

Time 1 3.51c 7.03±0.01b 7.17a Depth 0-15 cm Time 2 3.02±0.01c 6.89b 7.11±0.02a Time 3 2.63±0.11b 7.52±0.04a 7.48±0.02a Time 1 3.41±0.08a 2.89c 3.27±0.01b Depth 15-30 cm Time 2 3.03±0.01c 2.77±0.06b 3.14±0.03a Time 3 2.65c 4.78±0.04a 3.77±0.25b Time 1 3.39±0.01a 2.76±0.02c 2.85±0.01b Depth 30-45 cm Time 2 3.04±0.01a 2.64±0.06c 2.99±0.02b Time 3 2.58c 3.42±0.04a 3.17±0.01b CEC S SCBP STBP Time 1 0.84±0.05c 41.0±2.06a 23.4±1.45b Depth 0-15 cm Time 2 3.68±0.04c 34.7±1.07a 31.0±1.17b Time 3 10.7±0.47b 29.9±0.98a 29.1±0.96a Time 1 0.94±0.02c 10.4±0.33a 2.85±0.77b Depth 15-30 cm Time 2 3.06±0.49b 6.75±1.86a 4.43±0.06ab Time 3 7.95±1.39b 13.6±0.63a 10.0±0.83b Time 1 1.09±0.01c 10.1±0.76a 8.23±0.41b Depth 30-45 cm Time 2 3.23±0.16b 8.16±3.27a 6.11±0.34a Time 3 15.6±0.47a 1.64±0.14b 0.93c

Means±SD. For each row, different letters in different samples means significant differences (n=3, P < 0.05).

3.3. Evolution of Total Carbon (TC) and Nitrogen (TN)

At 0-15 cm depth, at times 1 and 2, the treatment SCBP had the significantly highest TC concentration, but at time 3, STBP had the highest TC content (Fig. 3A). At 15-30 and 30-45 cm, SCBP had significantly higher TC concentration than both S and STBP over time (P < 0.05)

(Fig. 3B and 3C). Total nitrogen was only detected at 0-15 cm depth and only in the amended soils (SCBP and SCTP). At all times, SCBP had the highest TN concentration (Fig. 4).

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Fig. 3. Evolution of the total carbon (TC) at three depths and at 3, 7 and 11 months of experiment (Time 1, 2 and 3, respectively). For each row, differ letters in different samples means significant differences (n=3, Student’s-t test P < 0.05). Error bars represent standard deviation.

Fig. 4. Evolution of the total nitrogen (TN) at depth 1 and at 3, 7 and 11 months of experiment

(Time 1, 2 and 3, respectively). For each time, differ letters in different samples means significant differences (n=3, Student’s-t test P < 0.05). Error bars represent standard deviation.

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3.4. Evolution of nutrients

At 0-15 cm depth, SCBP had the highest Ca, K, Mg and Na concentrations at time 1 (Table 4).

At time 2, SCBP had also the highest Ca and K concentrations, but Mg and Na were higher in

STBP. At time 3, both SCBP and STBP had the highest Ca and Na concentrations. The highest

K concentrations were detected in SCBP, and Mg in STBP (P < 0.05).

Table 4. Evolution of nutrients concentration (mg kg-1) in three depths and at 3, 7 and 11 months of experiment (Time 1, 2 and 3, respectively).

S SCBP STBP Ca 9.51±0.12c 4641±31.5a 2771±63.4b

K 9.18±0.07c 2572±146a 991±62.7b Mg 12.0±2.66c 790±40.7a 581±35.9b

Time 1 Time Na 25.7±2.20c 956±60.6a 458±37.5b

Ca 30.6±0.31c 4529±137a 3607±135b

15 cm 15

- K 1.68±0.69c 1262±44.2a 790±32.6b Mg 128±1.10c 768±23.0b 904±39.9a

Time 2 Time

Depth 0 Depth Na 22.3±0.14c 510±16.8b 701±17.4a Ca 94.8±4.29b 4639±151a 4441±148a

K u.l. 432±17.5a 225±5.42b Mg 496±24.1c 614±19.0b 704±22.8a

Time 3 Time Na 14.2±0.92b 115±4.96a 110±3.74a Ca 6.47±0.14c 329±40.4a 138±4.10b

K 8.30±0.87c 130±1.39a 58.1±2.24b Mg 8.64±0.56c 335±17.8a 82.3±3.44b

Time 1 Time Na 21.2±1.21c 70.0±1.81a 50.3±1.42b

Ca 28.8±4,60b 178±32.4a 98.6±1.25a

30 30 cm

- K 1.59±0.70b 10.1±5.41b 33.7±4.56a Mg 1.59±18.6b 10.1±50.8b 33.7±1.38a

Time 2 Time Na 20.6±0.54b 47.2±0.50a 53.9±8.62a

Depth 15 Depth Ca 89.8±2.43b 538±78.8a 115±26.3b

K u.l. 30.3±4.55a u.l. Mg 452±4.74a 49.4±3.63b 25.3±2.98c

Time 3 Time Na 7.95±1.39b 13.6±0.63a 10.0±0.83b Ca 7.01±0.18b 113±11.3a 118±0.42a

K 7.50±0.19c 19.6±1.80b 63.1±2.30a Mg 13.9±0.03c 343±23.3a 231±9.13b

Time 1 Time Na 20.8±0.62b 13.8±1.79c 33.3±1.11a

Ca 55.4±1.79b 159±33.1a 75.9±0.47ab

45 45 cm

- K 1.00±0.37c 9.95±3.59b 14.1±1.07a 30 Mg 102±4.47c 288±70.5a 187±13.3b

Time 2 Time Na 19.9±1.36b 44.6±1.76a 37.5±5.05a

Depth Depth Ca 90.4±2.40a 32.8±3.59b 16.8±0.032c

K u.l. u.l. u.l. Mg 606±18.5a 60.0±5.03b 26.6±0.07c

Time 3 Time Na 8.67±1.23a 7.72±0.45a 8.39±0.11a

Means±SD. For each row, different letters in different samples means significant differences (n=3, P < 0.05).

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At 15-30 cm, SCBP had the highest concentration of all nutrients at time 1 (Table 3). At time 2,

SCBP and STBP had higher Ca and Na concentrations than S, but STBP had the highest K and

Mg concentrations. At time 3, S had significantly higher Mg concentration than the amended

soils, but SCBP had the highest Ca, K and Na concentrations.

At 30-45 cm, both SCBP and STBP had the highest Ca concentrations at time 1 (Table 3). The

highest K and Na concentrations were shown by STBP, and SCBP had the highest Mg

concentrations (P < 0.05). At time 2, SCBP had the highest Ca and Mg concentrations, and both

STBP and SCBP had the highest Na concentrations. The highest K concentrations were

observed in STBP. At time 3, S had higher Ca and Mg concentrations than the amended soils.

Sodium concentrations showed no significant differences (P < 0.05) and K had undetectable

levels in all samples.

3.5. Harvested biomass of Brassica juncea L.

The mustard plants (Brassica juncea L.) were not capable of growing in the settling pond soil

(S), for this reason is not represented in Figure 5. B. juncea showed the highest dry biomass in

SCPB over time (Fig. 5A; P < 0.05). The height of B. juncea was higher in STBP at time 1 and

time 2, but at time 3 the height was the higher in SCBP (Fig. 5B, P < 0.05).

Fig. 5. Harvested biomass and height of Brassica juncea L. at 3, 7 and 11 months of experiment (Time 1, 2 and 3, respectively). For each time, different letters in different samples means significant differences (n=3, Student’s-t test P < 0.05). Error bars represent standard deviation

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4. Discussion

4.1. Effect of compost and technosol enhanced with biochar on soil carbon and nitrogen concentrations

Both compost mixed with biochar and technosol mixed with biochar significantly increased the total carbon concentration in the settling pond soil (Fig. 3). This was expected since the three compounds were rich in carbon (Table 2). This addition of C is very important for the reclamation of mine soils because, if the concentration of organic carbon increases, the possibility of retain available water and nutrients in bioavailable form also increases, and the structure of the soil and other physical properties improve (Lal, 2006). Moreover, the type of carbon in the biochar is highly recalcitrant, which means that it is not quickly mineralized and is stocked in the soil for long time (IBI, 2016).

Three months after starting the experiment, soil + compost + biochar (SCBP samples) had higher total carbon concentration in the first 15 cm than technosol enhanced with biochar

(STBP), because the used compost had higher TC concentration than the technosol (Table 2 and

Fig. 3). Eleven months after planting Brassica juncea L., in both SCBP and STBP carbon concentration was higher in the surface of the soils with technosol than in the soils with compost. One of the reasons is that carbon was leached from the soil surface faster in the samples with compost than in the samples with technosol, since it was observed an increase of

TC in depth in the SCBP samples.

Total nitrogen concentrations also significantly increased in the settling pond soil with the addition of compost and technosol together with biochar (Fig. 4), since the mine soil had undetectable concentrations of this nutrient and the three organic materials were rich in nitrogen

(Table 2). By comparing compost with technosol, the first increased more the TN concentration in the soil surface, since it had higher TN concentration (Table 2). That nitrogen added with the amendments was not leached for more than 15 cm depth, since the concentrations from 15 to 45 cm were always undetectable. Mustard plants reflected the higher concentration of nitrogen in the SCBP samples, since plant biomass was always the highest in the soils treated with compost

+ biochar (Fig. 5). This increase in soil TN due to the application of amendments is very

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Anexo IV important because nitrogen is a nutrient essential for plants and can be a limitation for their survival and growth if it is in low concentration (Christensen, 2004).

4.2. Effect of compost and technosol enhanced with biochar on other nutrients

Both compost and technosol, mixed with biochar, significantly increased the concentration of

Ca, K, Mg and Na in the mine soil (Table 4), since the three organic materials had higher concentration of all these nutrients before being added to the soil (Table 2).

Three months after starting the experiment, the samples with compost showed the highest concentration of those nutrients in the soil surface (Table 4), although the used technosol had higher amount of them than the compost before being added (Table 2). This could indicate that there was higher concentration of Ca, K, Mg and Na in available form for plants in technosol than in compost, and that higher amount of these nutrients was taken up by mustard plants in the

STBP than in STBP, during the first three months. Another reason is that Ca, K and Na were also leached faster in the samples with technosol, as the concentration of K and Na were the highest in STBP at 30-45 cm depth, and Ca concentrations were similar to the SCBP samples.

Eleven months after starting the experiment, the samples amended with technosol + biochar had the highest concentration of Mg in the surface (0-15 cm), as well as Ca and Na concentrations similar to the SCBP samples. In the case of Ca and Mg at STBP samples, as we observed a concentration higher at 11 months than at 3 months, it is possible that this nutrient was released from non-exchangeable forms, which suggest a high potential of technosol for provide calcium to plants. In the case of Na, it was probably leached and taken up by plants.

4.3. Principal component analysis (PCA) of the samples

The values of TC, TN, Ca, K, Mg and Na were selected to perform a PCA (Table 4). The PCA was performed separately for each sampling time (Table 5). The soil samples were located in a scatter plot based on the results from the PC obtained (Fig. 6). None of the samples is in the same quadrant, indicating that the two types of amendments significantly changed the concentrations of carbon, nitrogen and the other studied nutrients during the whole time of the experiment.

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Table 5. The component score coefficients matrix from the PCA for the three sampling times

Indicators PC1 PC2 TC 0.45 0.89 TN 0.69 0.71 Time 1 Ca 0.75 0.66 K 0.88 0.46 Mg 0.64 0.76 Na 0.84 0.53 TC 0.73 0.67 TN 0.79 0.59 Time 2 Ca 0.81 0.58 K 0.90 0.42 Mg 0.55 0.83 Na 0.45 0.88 TC 0.62 0.76 TN 0.85 0.52 Ca 0.72 0.68 Time 3 K 0.95 0.29 Mg 0.30 0.94 Na 0.72 0.68

Fig. 6. Scatter plot with the two principal components obtained in the PCA (PC 1 and PC 2) referred to the soil samples with the different treatments.

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For time 1, two principal components (PC1 and PC2) were obtained that account for 99.92% of the total variance. PC1 and PC2 explain 95.9% and 4% of the variance, respectively. According to both the rotated component matrix (Table 5) and the position of the samples on the scatter plot (Fig. 6), after 3 months of experiment (time 1) the SCBP significantly increased total carbon and nitrogen as well as Ca, K, Mg and Na. The STBP samples also increased TC and TN and then Ca and Mg, but did not increase K and Na.For time 2, PC1 and PC2 were obtained and account for 99.90% of the total variance. PC1 and PC2 explain 95.1% and 4.8% of the variance, respectively. For time 3, PC1 and PC2 were obtained and account for 99.70% of the total variance. PC1 and PC2 explain 91.8% and 7.8% of the variance, respectively. For time 2 and 3, the distribution of the samples in the scatter plot was the same (Fig. 6). After seven months of experiment (time 2), the samples with compost significantly increased TC and TN, Ca and K.

The samples with technosol increased TC, Mg and Na. After eleven months of experiment, the compost-samples increased all the studied nutrients except Mg, and the technosol-samples all except total nitrogen and K.

5. Conclusions

The addition of a technosol made of wastes or a compost, both mixed with biochar, to a poor mine soil, significantly increased the concentration of soil total carbon and nitrogen. Three months after the application of the amendments, the mixture with compost increased carbon concentration more than the mixture with technosol but, at 11 months, the concentrations were higher in the technosol. That indicates that the used technosol had forms of carbon more stable than compost, which could be released slower than in the compost-amended soils. Total nitrogen was always higher in the samples with compost. We detected that nitrogen was not leached beyond 15 cm depth, which means that the used amendments maintain that nutrient in the soil surface, where usually the highest root density is located.

Both compost and technosol mixed with biochar also increased the concentration of calcium, potassium, magnesium and sodium in exchangeable form in the mine soil. Since these nutrients were in lower concentration with technosol than with compost 3 months after the amendment, in

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7. References

AEMET, 2016. Valores Climatológicos Normales. Santiago de Compostela Aeropuerto.

http://www.aemet.es/es/serviciosclimaticos/datosclimatologicos/valoresclimatologicos?l=1

428&k=gal.

Beesley, L., Moreno-Jiménez, E., Gomez-Eyles, J.L., Harris, E., Robinson, B., Sizmur, T.,

2011. A review of biochars’ potential role in the remediation, revegetation and restoration

of contaminated soils. Environ. Pollut. 159, 3269–82. doi:10.1016/j.envpol.2011.07.023

Christensen, B.T., 2004. Tightening the Nitrogen Cycle, in: Schjonning, P., Elmholt, S.,

Christensen, B.T. (Eds.), Managing Soil Quality, Challenges in Modern Agriculture. CABI

Publishing, London, UK, pp. 44–66.

Conesa, H.M., Faz, Á., Arnaldos, R., 2006. Heavy metal accumulation and tolerance in plants

from mine tailings of the semiarid Cartagena–La Unión mining district (SE Spain). Sci.

Total Environ. 366, 1–11. doi:10.1016/j.scitotenv.2005.12.008

Gandolfi, I., Sicolo, M., Franzetti, A., Fontanarosa, E., Santagostino, A., Bestetti, G., 2010.

Influence of compost amendment on microbial community and ecotoxicity of

hydrocarbon-contaminated soils. Bioresour. Technol. 101, 568–575.

doi:10.1016/j.biortech.2009.08.095

Huang, M., Zhu, Y., Li, Z., Huang, B., Luo, N., Liu, C., Zeng, G., 2016. Compost as a Soil

Amendment to Remediate Heavy Metal-Contaminated Agricultural Soil: Mechanisms,

Efficacy, Problems, and Strategies. Water, Air, Soil Pollut. 227, 359. doi:10.1007/s11270-

016-3068-8

IBI, 2016. International Biochar Initiative [WWW Document]. URL http://www.biochar-

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international.org/

IUSS Working Group WRB, 2014. World Reference Base for Soil Resources 2014.

International soil classification system for naming soils and creating legends for soil maps.

Rome, Italy.

Lal, R., 2006. Enhancing crop yields in the developing countries through restoration of the soil

organic carbon pool in agricultural lands. L. Degrad. Dev. 17, 197–209.

doi:10.1002/ldr.696

Lehmann, A., Schad, P., 2007. Soils , Section 2 : Global change and environmental risk

assessment World Reference Base for Soil Resources.

Manzano, R., Silvetti, M., Garau, G., Deiana, S., Castaldi, P., 2016. Influence of iron-rich water

treatment residues and compost on the mobility of metal(loid)s in mine soils. Geoderma

283, 1–9. doi:10.1016/j.geoderma.2016.07.024

Parraga-Aguado, I., González-Alcaraz, M.N., Schulin, R., Conesa, H.M., 2015. The potential

use of Piptatherum miliaceum for the phytomanagement of mine tailings in semiarid areas:

Role of soil fertility and plant competition. J. Environ. Manage. 158, 74–84.

doi:10.1016/j.jenvman.2015.04.041

Porta, J., 1986. Técnicas y experimentos en Edafología. Collegi Oficial D`Enginyers Agronoms

de Catalunya, Barcelona, Spain.

Puga, A.P., Abreu, C.A., Melo, L.C.A., Beesley, L., 2015b. Biochar application to a

contaminated soil reduces the availability and plant uptake of zinc, lead and cadmium. J.

Environ. Manage. 159, 86–93. doi:10.1016/j.jenvman.2015.05.036

Puga, A.P., Melo, L.C.A., de Abreu, C.A., Coscione, A.R., Paz-Ferreiro, J., 2016. Leaching and

fractionation of heavy metals in mining soils amended with biochar. Soil Tillage Res. 164,

25–33. doi:10.1016/j.still.2016.01.008

Rodríguez-Vila, A., Asensio, V., Forján, R., Covelo, E.F., 2016. Chemical fractionation of Cu,

Ni, Pb and Zn in a mine soil amended with compost and biochar and vegetated with

Brassica juncea L. J. Geochemical Explor. 158, 74–81. doi:10.1016/j.gexplo.2015.07.005

Venegas, A., Rigol, A., Vidal, M., 2016. Changes in heavy metal extractability from

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7. Discusión general

Discusión general

7. Discusión general

En el capítulo 1 y anexo I se muestran los resultados de los efectos provocados por los tratamientos elaborados con tecnosol y biochar sobre el suelo de la balsa de la mina de Touro, haciendo hincapié en las concentraciones fitodisponible de metales y los contenidos de nutrientes. La evolución de las concentraciones fitodisponibles de metales y los contenidos de nutrientes en los tratamientos elaborados con compost y biochar se expusieron en el capítulo 2 y anexo II. Por último, en los anexos III y IV se expusieron los resultados donde se comparan los efectos de los tratamientos elaborados con technosol y biochar frente a los elaborados con compost y biochar. En la presente discusión general, se mostrará por separado el efecto de la adición de tecnosol y biochar y la revegetación con Brassica juncea L. sobre el suelo de la balsa de decantación de la mina de Touro (capítulo 1 y anexo I); el efecto de la adición de compost y biochar y la revegetación con Brassica juncea L. sobre el suelo de la balsa de decantación de la mina de Touro (capítulo 2 y anexo II), y por último las comparativa de los tratamientos elaborados con tecnosol y biochar frente a los elaborados con compost y biochar ambos revegetados con Brassica juncea L. (anexos III y IV).

7.1 Efecto de la adición de tecnosol y biochar y de la revegetación con Brassica juncea L. sobre el suelo de la balsa de decantación de la mina de Touro (Capítulo 1 y Anexo I)

7.1. 1 Evolución del pH

El suelo de la balsa de decantación (S) se clasifica como extremadamente ácido según su valor de pH (Hazelton y Murphy, 2007). El bajo pH de este suelo se debe sobre todo a sus contenidos en minerales sulfurosos los cuales, en contacto con el agua y el aire, producen ácido sulfúrico y, por otro lado, dan lugar al fenómeno denominado drenaje ácido de mina (Gomes et al., 2016, Pataca, 2004).

En la profundidad 0-15cm, tanto la aplicación de tecnosol como de tecnosol combinado con biochar (con y sin planta) aumentaron el pH del suelo de la balsa de decantación (S) (Figura 3, anexo I). Esto es probablemente debido a los elementos con los que se elaboraró el tecnosol como, por ejemplo, las cenizas (Hu et al., 2014) o los lodos de depurada (Alvarenga et al., 2016) y al biochar, ya que, en general, los biochar suelen tener un pH muy alto (Ibrahim et al., 2016, Rehman et al., 2016). Si analizamos el comportamiento de los cuatro tratamientos en la profundidad 0-15 cm, se observa como los elaborados con tecnosol y biochar aumentaron el pH de forma significativa en comparación con la aplicación directa de tecnosol (Figura 3, anexo I), observándose, por un lado, como ya demostró Fowles (2007), que el biochar puede potenciar los

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Discusión general efectos positivos de enmiendas elaboradas con residuos y, por otro lado, como concluyeron Karer et al. (2015) y Zhang et al. (2013), que la aplicación de biochar al suelo aumenta el pH.

En las profundidades 15-30cm y 30-45 cm, el aumento de pH es progresivo, siendo más pronunciado en el último tiempo (Figura 3, anexo I). Como ocurría en la profundiad 0-15 cm, los tratamientos STB y STBP fueron los de mejor efecto sobre la acidez del suelo.

En un suelo tan ácido, el aumento de pH es importante, siendo un factor crucial a la hora de reducir las concentraciones fitodisponibles de elementos potencialemente tóxicos (Cui et al., 2016, Gusiatin et al., 2016). Esta relación entre pH y la concentración fitodisponibles de metales quedó reflejada con la existencia de correlaciones negativas de Pearson con unos valores de r=- 0,88, r=0-,94, r=-0,65 para el Cu, Ni y Pb respetivamente (P˂0,01); el Zn fue la excepción con una correlación positiva (r=943, P˂0,01). Autores como Houben et al. (2013) ya observaron una correlación entre el aumento de pH y la disminución de las concentraciones fitodisponibles de ciertos metales, argumentando que el aumento de pH del suelo induce la inmovilización de los metales porque favorece la precipitación, disminuye la solubilidad y promueve la adsorción de los metales debido al aumento de la carga negativa de los componentes suelo. Por otro lado, autores como Rodríguez-Vila. (2016) concluyeron que la aplicación de tecnosol y biochar aumenta el pH y reduce la movilidad Cu, Ni y Pb.

Además, el pH es un factor determinante para la restauración de suelos degradados debido a que juega un papel crítico en la revegetación, lo cual detallaremos en un apartado posterior, influyendo en la mejora de la disponibilidad de nutrientes y la supervivencia de las plantas durante las primeras etapas de desarrollo durante el establecimiento de vegetación (Reverchon et al., 2015, Shrestha and Lal, 2011; Zhang et al, 2013). Por lo tanto, la aplicación tanto de tecnosol como de tecnosol combinado con biochar ejerce un efecto positivo al aumentar los valores de pH lo cual mejora la calidad del suelo de la balsa de decantación.

7.1.2 Evolución del contenido total de carbono total (CT)

Uno de los problemas más importantes y generalizados a la hora de recuperar un suelo de mina es su bajo contenido en materia orgánica (Zhou et al., 2015). Esto también ocurre en el suelo de la balsa de decantación de la mina de Touro (Figura 4, anexo I). En la profundidad 0-15 cm, se observa como todos los tratamientos aplicados tienen un efecto positivo debido al carbono aportado por el tecnosol y el biochar (Figura 4, anexo I) (Vidal-Beaudet et al., 2012, Biederman y Harpole, 2013). La aplicación de la combinación de tecnosol y biochar provocaron un mayor aumento de CT. Este acusado incremento del CT provocado por el biochar ya fue estudiado por autores como Biederman y Harpole (2013) y Madiba et al. (2016). El biochar aporta formas de carbono altamente recalcitrantes, lo cual es muy interesante a la hora de recuperar un suelo

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Discusión general altamente degradado (IBI, 2015). Este aporte de carbono orgánico es importante ya que afecta positivamente a reducir la concentración de metales fitodisponibles, incrementar la capacidad de intercabio catiónica, el pH, la capacidad de retención de agua disponible en el suelo, etc. (Karer et al., 2015; Rodríguez-Vila et al., 2015, Puga et al., 2015).

Los tratamientos aplicados también fueron efectivos a la hora de aumentar el CT en las profundidades 15-30 cm y 30-45cm (Figura 4, anexo I). En los dos primeros tiempos se observó un mismo patrón, en el cual los tratamientos elaborados con tecnosol y biochar (con y sin planta) fueron los que más CT aportaron. Este mayor aumento es debido al tipo carbono aportado por el biochar, como demostraron Darby et al. (2016), los cuales concluyeron, después de comparar diferentes tratamientos (con y sin biochar y solo biochar) aplicados a un suelo, que cuando se aplica solo biochar aumenta el carbono en comparación con los otros tratamientos. Por otro lado, Liu et al. (2016) expusieron que la aplicación de biochar también aumenta el contenido de materia orgánica del suelo, el agua extraíble, carbono orgánico en el suelo y la concentración de carbono orgánico en agua de poro y lixiviado. En nuestro caso, ésto solo se cumplió en el primer y segundo tiempo ya que, en el último tiempo, este aporte de CT se iguala en los diferentes tratamientos incluso siendo mayor en algunos de los tratamientos sin biochar.

-1 7.1.3 Evolución de la capacidad de intercambio catiónico (CEC) (cmol(+)kg ), saturación de bases (V%), y saturación de aluminio (Al%)

Los valores de CEC en el suelo de la balsa de decantación son muy bajos (Capítulo 2, Tabla 3). Esta baja CEC es un gran obstáculo a salvar a la hora de recuperar un suelo de este tipo, ya que una baja CEC indica una baja resistencia del suelo a la hora de afrontar los cambios que pueda sufrir el mismo (Hazelton y Murphy 2007). Por otro lado, la CEC está relacionada con la biodisponibilidad de metales (Chandra et al., 2014), por lo que es muy importante corregirla de forma correcta.

En la profundidad 0-15 cm, el incremento de la CEC fue significativo una vez aplicados tanto el tecnosol como el tecnosol con biochar (con o sin planta) (Tabla 3, anexo I). Este aumento de CEC se debe claramente a la influencia del tecnosol, ya que éste presentó una CEC de

-1 -1 76,0±4,80 (cmol(+)kg ) mientras que el biochar presentó una CEC de 15,8±17,8 (cmol(+)kg ). Aun así, ambas enmiendas (tecnosol y biochar) pueden provocar un aumento de la CEC (Asensio et al., 2013, Fellet et al., 2014), ya que, por un lado, el tecnosol está elaborado con residuos con alto contenido en cationes básicos (Tabla 2, anexo I) y, por otro lado, la estructura porosa del biochar le confiere una gran superficie específica, lo cual contribuye al aumento de la CEC del suelo cuando el biochar es aplicado sobre él (Ohsowski et al., 2012). Si calculamos el grado de saturación de bases (V%) y saturación de aluminio (Al%), se puede observar como V% en el

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Discusión general suelo pasa de ser de un 39,4% a un 99% una vez se le aplican los diferentes tratamientos (Tabla 3, anexo I). Además, en esta profundidad (0-15 cm), el Al% tiene un valor de 0 en el suelo tratado, lo cual demuestra que este aumento de CEC se debe al aporte de cationes básicos (Tabla 3, anexo I). Los tratamientos que combinaban tecnosol y biochar, al transcurrir el primer y segundo tiempo, presentaron una tendencia a tener una CEC mayor (Tabla 3, anexo I). Esto se debe posiblemente a la alta capacidad de retención de cationes por parte del biochar, inducida por su alta aromaticidad, área superficial específica y carga negativa (Karer et al., 2015, Puga et al., 2015).

El suelo de la balsa de decantación presentó los mayores valores de CEC en la profundidad 15-30 cm (Tabla 3, capítulo 2). Este alto valor de CEC es debido a un alto porcentaje de cationes ácidos, sobre todo al aluminio de cambio (62% de media). Este aluminio de cambio fue menor en el suelo una vez aplicados los diferentes tratamientos (ST, STP, STB y STBP) con valores en general inferiores al 50% (Tabla 3, anexo I). Por último, transcurridos 11 meses se observó como los tratamientos elaborados con tecnosol y biochar presentaron una mayor V% (72% de media) y un menor Al% (28 % de media) que los tratamientos elaborados solo con tecnosol. Estas diferencias nos indican la capacidad del biochar para retener cationes básicos (Karer et al., 2015, Puga et al., 2015).

En la profundidad 30-45cm, la aplicación de tecnosol combinado con biochar y vegetado con Brassica juncea L. (STBP) presentó el valor más alto de CEC en los dos primeros tiempos (Tabla 3, anexo I), pero también presentó un mayor Al% y una menor V%, por lo cual el contenido de cationes básicos fue menor que en el resto de tratamientos. Esta menor %V en STBP se debe posiblemente a la retención de cationes básicos por parte del biochar en las primeras profundidades (Karer et al., 2015, Puga et al., 2015). Sin embargo, en el tiempo 3 fue el suelo de la balsa sin tratatar el que presentó una mayor CEC. Al final del tiempo experimental en esta profundidad tanto el suelo sin tratar como los tratamientos presentaron una %Al con valores cercanos al 60% y una %V con valores cercanos al 40% (Tabla 3, anexo I). Analizando los valores de la %V durante el tiempo experimental observamos como a partir del segundo tiempo estos valores están por debajo de 50% en todos los tratamientos aplicados, lo que nos indica que los tratamientos dejan de tener influencia y se corta el flujo de cationes básicos. Hemos de tener en cuenta que en esta última profundidad solo tenemos suelo de mina con un pH˂4 lo cual dificulta el efecto de los tratamientos después de 11 meses, de hecho, en nuestro experimento se observo una correlación de Pearson positiva entre la CEC y el pH (r=0,88; p˂0,01).

7.1.4 Evolución del contenido total de nitrógeno (NT) en la profundidad 0-15 cm

El contenido de nitrógeno, tanto en el suelo de la balsa de decantación sin tratar como en el tratado con arena (S y SS), fue indetectable en las tres profundidades estudiadas, es decir, sus

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Discusión general contenidos estaban por debajo del límite de detección NT <0.1% (Figura 5, anexo I). Este bajo contenido en NT es un factor limitante importante a la hora de intentar establecer una cubierta vegetal estable, limitación que detallaremos más adelante en un apartado específico. Está demostrado que, en un suelo de mina, una vez se le aplican enmiendas, mejoran los contenidos de nitrógeno y tiene más facilidad para desarrollar una cobertura vegetal (Zanuzzi et al., 2013). Todos los tratamientos aplicados aumentaron de forma significativa el contenido en nitrógeno debido, principalmente, al contenido de nitrógeno que presentaba el tecnosol (Tabla 2, anexo I). Los residuos con los que se elaboró el tecnosol (lodos de depuradora o restos de industrias agroalimentarias) poseen un contenido considerable de nitrógeno (Jordán et al., 2017, Zanuzzi et al., 2013). Además, esta mejora del contenido en nitrógeno es importante a la hora de recuperar y mejorar la actividad microbiana en el suelo (Asensio et al., 2013). Sin embargo, en las profundidades 15-30 cm y 30-15 cm el NT no fue detectable (Figure 5, anexo I), posiblemente debido a su retención en la profundidad 0-15 cm y a su fácil migración através de estas dos últimas profundidades.

7.1.5 Relación carbono y nitrógeno (C/N) en la profundidad 15 cm

En la figura 1 se muestra la relación entre el carbono y nitrógeno (C/N) de los tratamientos aplicados en la primera profundidad. La C/N del suelo de la balsa de decantación sin tratar y del control SS no se muestran debido a que su contenido en nitrógeno fue no detectable. Según Troeh y Thompson (2005) el rango óptimo de C/N para que el nitrógeno este mineralizado abarca desde C/N ˃10 a C/N ˂30. Todos los tratamientos aplicados conseguieron establecer una C/N dentro del rango óptimo en el suelo de la balsa de decantación para que el nitrógeno este en forma mineralizada.

Figura 1. Relación del contenido de carbon y nitrógeno (C/N) en la profundidad 0-15 cm.

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Discusión general

- + El nitrógeno en forma mineralizada, en forma de nitrato (NO3 ) y el amonio (NH4 ), es el que puede ser absorbido por las plantas. A lo largo de los 11 meses de experimento los valores de C/N en los tratamientos se mantuvieron dentro del rango óptimo, ésto es importante, ya que de lo contrario el nitrógeno estaría inmovilizado y no podría ser absorbido por las plantas (Troeh y Thompson, 2005).

Los tratamientos que combinaban tecnosol y biochar presentaron una C/N mayor que la aplicación directa de tecnosol. Esto se debe a que el biochar aumenta mucho los valores de carbono en el suelo, lo que provoca el aumento de la relación entre el carbono y nitrógeno.

7.1.6 Factores limitantes para la producción vegetal en la profundidad 0-15 cm

En este trabajo se estudiaron ocho factores limitantes para la producción vegetal los cuales detallaremos acontinuación (Tabla 5, anexo I): el Factor c está relacionado con el pH. Según este factor, si un suelo presenta un pH<3,5 la vegetación tendría dificultades para desarrollarse sobre él. El factor relacionado con la deficiencia de Ca es el Factor Ca, se considera que un suelo es

-1 deficitario en Ca cuando su valor del complejo de cambio es Ca<1.5cmol(+)kg . Según el Factor

-1 e, los valores mínimos de CEC para el asentamiento de vegetación serían CEC <4 cmol(+)kg .

-1 Según el Factor K, un suelo con valores de K por debajo de K <0,2 cmol(+)kg estaría limitado por el K para la producción vegetal. El Factor Mg es el referente a la deficiencia de Mg, se aplica a los suelos en los que el nivel de Mg asimilable no es el óptimo para las plantas. Un suelo se

-1 considera afectado por este factor cuando su contenido en Mg es de Mg <0,4 cmol(+)kg . El Factor n se estima positivo cuando la saturación de Na en el complejo de cambio de los 50 cm superficiales es Na≥ 15%. Por último, el Factor N, presencia de valores de nitrógeno total por debajo de <0,1%, valor a partir del cual la mayoría de las plantas no podrían desarrollarse

El suelo de la balsa de decantación sin tratar y el tratado con arena (S y SS) están afectados como mínimo por 4 de los ocho factores limitantes evaluados a lo largo de todo el experimento. El suelo S se vio afectado por el Factor c, Factor Ca, Factor K, Factor N durante todo el experimento. En cuanto al Factor e, relativo a la CEC, aunque solo aparece afectado en el primer tiempo hemos de recordar que, aunque dicho valor aumente, es debido sobre todo al porcentaje de Al+3 de cambio en este caso (Tabla 5, anexo I).

Una vez se aplicaron los tratamientos sobre el suelo de la balsa de decantación, éste dejo de estar afectado por el Factor c (Tabla 5, anexo I). Hemos de recordar que, con el transcurso del tiempo, se alcanzaron valores de pH óptimos para el crecimiento de la mayoría de las plantas. Los valores más altos del Factor c se obtuvieron en los tratamientos con biochar (STB y STBP) debido a la alta capacidad del biochar para aumentar el pH (Hazelton y Murphy, 2007; Ohsowski et al., 2012).

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Discusión general

El aumento de los valores del Factor Ca es muy importante ya que nos indica que la biodisponibilidad de calcio para las plantas aumenta, lo cual se debe posiblemente a la mejora de las condiciones de pH (Hazelton y Murphy, 2007). Una vez tratado el suelo de la balsa de decantación, el valor crítico del Factor Ca es superado durante los 11 meses en los que se llevó a cabo el experimento (Tabla 5, anexo I).

Según diferentes autores como Buol et al. (1975) y Macías y Calvo (1983), el suelo de la balsa de decantación sólo se vería afectado por el Factor e al inicio del experimento (Tiempo 1, Tabla 5, capítulo 2), pero autores como Hazelton y Murphy (2007) consideran que los suelos con una CEC˂6 tambien están afectados por el Factor e. Un valor bajo de CEC indica una baja resistencia a los cambios en la química del suelo que son causados por el uso del mismo. Por ello, el aumento de CEC producido por los tratamientos es importante ya que es un factor importante para que la vegetación sea capaz de establecerse en el suelo objeto de estudio.

Todos los tratamientos consiguieron aumentar del Factor K hasta superar considerablemente el límite establecido por Buol et al. (1975) y Macías y Calvo (1983) (Tabla 5, capítulo 2). En el caso del Factor K cabe destacar que los valores fueron mucho más altos en el primer tiempo y luego fueron disminuyendo, siempre con valores queduplicaban los establecidos como límite para dicho factor.

Una vez aplicado el tecnosol solo o combinado con biochar (con y sin planta) aumentó el contenido de nitrógeno, lo que se reflejó en que el suelo de la balsa de decantación dejase de estar afectado por el factor Factor N. Este aumento en los contenidos de nitrógeno mediante la aplicación de enmiendas es importante para mejorar la calidad de suelos degradados y, además, su importancia es notoria a la hora de intentar establecer una cobertura vegetal estable en este tipo de suelos, aspectos en los que ya trabajaron investigadores como Christensen, (2004) o Zanuzzi et al., (2013).

El único factor de los estudiados por el que se verían afectados los distintos tratamientos sería por el Factor n al no conseguir aumentar el porcentaje de Na hasta el 15%, valor límite para que un suelo no se considere afectado por este factor.

7.1.7 Evolución de la biomasa cosechada de las Brassica juncea L. cultivadas

Las Brassica juncea L. cultivadas no crecieron en ninguno de los controles (S, SS) debido a las carencias de nutrientes, bajo pH y altas concentraciones fitodisponibles de los metales estudiados (Figura 6, anexo I). Sin embargo, las Brassica juncea L cultivadas sobre el tecnosol y el tecnosol combinado con biochar, se establecieron y desarrollaron a lo largo de todo el tiempo experimental, quedando patente el efecto positivo de estos tratamientos sobre la implantación de una cubierta vegetal en suelos degradados. Analizando los datos de biomasa y altura de las

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Discusión general

Brassica juncea L., las plantas cultivadas en el tratamiento elaborado con tecnosol y biochar presentaron una mayor biomasa y altura y, por lo tanto, vigorosidad a lo largo del experimento (Figura 6, anexo I).

Este mayor crecimiento de las Brassica juncea L. en el suelo tratado con tecnosol y biochar es importante ya que, si recordamos los datos de NT en el tratamiento STBP, en el tercer tiempo presentaba el menor contenido de NT (Figura 6, anexo I) pero, sin embargo, en este tiempo, este tratamiento presentó la mayor biomasa cosechada. Esto es debido a que el biochar mejora la eficiencia del uso de N y P (Zhu et al., 2014). El biochar contiene materia orgánica aromática estable con altas concentraciones de carbono de 70-80% y materia mineral, incluyendo nutrientes. Además, la alta área superficiar del biochar que le confiere su alta porosidad unida a la carga variable y grupos funcionales que pueden aumentar la capacidad de retención de agua en el suelo, la capacidad de intercambio catiónico (CEC) y la capacidad de sorción superficial (Anawar et al., 2015). Esto provoca que se reduzca la migración de nitratos y que las plantas puedan utilizar los nutrientes con mayor eficacia (Beesley y Marmiroli, 2011). El biochar, debido a sus características, puede provocar resistencia de los cultivos a las enfermedades lo que se trasmite en un mejor crecimiento de los mismos (Anawar et al., 2015).

7.1.8 Evolución de las concentraciones fitodisponibles de Cu, Pb, Ni, Zn

En la profundidad 0-15 cm, los tratamientos provocaron un efecto positivo en las concentraciones fitodisponibles de los metales (Cu, Ni, Pb, Zn) reduciendo las de Cu, Pb y Ni, aunque no las de Zn, en comparación con el suelo de la balsa de decantación sin tratamiento (Tabla 3.1, capítulo 1). La aplicación directa de tecnosol presentó las mayores concentraciones fitodisponibles de Zn debido a los residuos con los que se elaboró el tecnosol. Estas concentraciones fitodisponibles de Zn suelen aparecer en el suelo cuando se le aplican residuos como lodos de depuradora o restos de industrias agroalimentarias los cuales suelen contener altas concentraciones de Zn (Alvarenga et al., 2016, González-González et al., 2013). Dentro de los tratamientos aplicados, el tratamiento que combina tecnosol con biochar y Brassica juncea L (STBP) fue el más efectivo en este aspecto ya que, al final de los 11 meses, presentó menores concentraciones fitodisponibles de tres (Cu, Ni y Zn) de los cuatro metales estudiados (Cu, Pb, Ni, Zn).

Como ya ha sido comentado, los tratamientos provocaron un aumento de los valores de pH, CT y CEC en el suelo de la balsa. La reducción de las concentraciones fitodisponibles de metales está relacionada directamente con el aumento de estos factores (Cui et al., 2016, Gusiatin et al., 2016, Chandra et al., 2014, Karer et al., 2015; Puga et al., 2015).

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Discusión general

En la profundidad 15-30 cm (Tabla 3.2, capítulo 1), si comparamos los datos de porcentaje de concentraciones fitodisponibles frente a las concentraciones totales al final del tiempo experimental (Tabla 1), se observa como las enmiendas sí son efectivas al disminuir este porcentaje, salvo la excepción del Cu en los casos donde el tecnosol fue aplicado directamente. También se puede observar como, en líneas generales, la aplicación de tecnosol combinada con biochar en comparación con la aplicación directa de tecnosol presentó menor porcentaje de las concentraciones fitodisponibles frente a las concentraciones totales. De esta forma vuelve a quedar patente el efecto positivo de combinar el biochar con este tipo de enmiendas.

Después de los once meses de experimento se observó, que en la profundidad 30-45cm, tanto la aplicación de tecnosol como de tecnosol combinada con biochar (con y sin planta) es efectiva a la hora de disminuir las concentraciones fitodisponibles de metales en el suelo de la balsa de decantación (S) (Tabla 3.3, capítulo 1), con la excepción del Cu en el suelo tratado unicamente con tecnosol, sin diferencias significativas en comparación con S. Tenemos que recordar que los tratamientos son aplicados en superficie y, esta profundidad del cilindro (30- 45cm) está formada por suelo de la balsa de decantación y las partículas que hayan podido migrar desde las enmiendas. En esta profundidad (30-45 cm), algo que destacó al final de los 11 meses de experimento, fue el efecto del tratamiento que combina tecnosol conbiochar y Brassica juncea L. (STBP), el cual presentó menores concentraciones fitodisponibles de todos los metales en comparación con S y menores concentraciones fitodisponibles de Cu, Ni y Zn en comparación con los tratamientos elaborados solo con tecnosol.

Tabla 1. Profundidad 15-30 cm, tiempo 3. Porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn.

% S SS ST STP STB STBP

Cu 11,4 12,4 12,6 13,2 9,65 9,81

Pb 1,34 1,50 1,11 1,22 0,90 1,21

Ni 28,7 22,2 19,1 17,7 17,4 13,9

Zn 9,56 6,05 7,61 7,43 7,49 6.83

7.1.9 Evolución de los contenidos cosechables de Cu, Pb, Ni, Zn, Coeficiente de Trasferencia (TrC) y Factor de Translocación (TF)

7.1.9.1 Contenidos cosechables de Cu, Pb, Ni, Zn.

En los tallos y raíces de las Brassica juncea L. cultivadas sobre la combinación de tecnosol y biochar, los contenidos cosechados de Cu, Pb, Ni y Zn fueron, en líneas generales,

279

Discusión general superiores en comparación con los contenidos de las Brassica juncea L. cultivadas sobre la aplicación directa de tecnosol (Figura 4, capítulo 1). El metal cosechado en mayor cantidad fue el Zn, posiblemente debido a que fue el metal con mayores concentraciones fitodisponibles en el material de partida (Tabla 2, capítulo 1) y a que el Zn es el metal con menor afinidad por el tipo de enmiendas con las que se elaboró este tratamiento (tecnosol principalmente) (Forján et al., 2016a y b). El biochar es un acondicionador del suelo que mejora el crecimiento de las plantas al suministrar y retener nutrientes, y al proporcionar otros beneficios como mejorar las propiedades físicas, químicas y biológicas del suelo (Fellet et al., 2011). Posiblemente debido a las características citadas anteriormente del biochar, las Brassica juncea L. cultivadas en STBP hayan presentado mayores contenidos de Cu, Pb, Ni y Zn al final del tiempo experimental. Además, si observamos los datos de las concentraciones fitodisponibles de los metales estudiados en la zona de mayor acción de las raíces (0-15 cm), coincide que el tratamiento STBP presentó, en prácticamente todos los casos, concentraciones fitodisponibles significativamente inferiores en comparación con el tratamiento STP (Figura 3, capítulo 1). Autores como Prapagdee et al. (2014) ya han demostrado que la aplicación de biochar en suelos contaminados por metales para mejorar el crecimiento de las plantas también puede conducir a un aumento en la absorción de metales por algunas plantas.

7.1.9.2 Evolución de el Coeficiente de tranferencia (TrC) en las Brassica juncea L. cultivadas sobre STP y STBP

Busuioc et al. (2011) propusieron que, para que una planta se considere fitoextractora, debe presentar valores mayores que 1 del coeficiente de tranferencia (TrC˃1). Una vez calculado el TrC para los metales estudiados en este trabajo (Cu, Ni, Pb y Zn) se observó que, a lo largo del tiempo y tanto en el tratamiento STP como en el tratamiento STBP, los valores de este coeficiente no superaban el 0,9 (Figura 6, capítulo 1). Esto nos indica que, en nuestro caso, las Brassica juncea L. no presentan capacidad fitoextractora. Si embargo, según Kidd et al. (2009), las plantas efectivas a la hora de fitoestabilizar tienen coeficientes de transferencia raíz-tallo bajos, como ocurre en nuestro caso. Por lo tanto, nuestras Brassica juncea L. presentan un claro papel fitoestabilizador de los metales estudiados. Además, hace décadas, autores como Cunningham et al. (1995) concluyeron que la fitoestabilización es la técnica más adecuada para materiales relativamente inmóviles y grandes superficies, y es aceptable para la recuperación de suelos de mina.

7.1.9.3 Evolución de el Factor de translocación (TF) en las Brassica juncea L. cultivadas sobre STP y STBP

Para que una planta se considere que tiene capacidad de translocar metales de la raíz al tallo, según el valor del factor de translocación (TF), éste debe ser mayor que 1 (TF˃1) (Baker y

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Discusión general

Brooks, 1989). Sin embargo, en nuestro caso, solo a los 11 meses y en las Brassica juncea L. cosechadas en el tratamiento que combinaba tecnosol ybiochar (STBP), este factor supera el valor de 1 para el Pb y el Zn (Figura 5, capítulo 1). Esto sugiere que Brassica juncea L. puede ser una buena especie translocadora para Pb y Zn una vez que el tratamiento STBP se ha estabilizado. Por otro lado, que los valores de TF para Cu y Ni no haya superado el valor de 1 no significa que para estos metales las Brassica juncea L. no hayan sido efectivas, ya que según Nouri et al. (2009) que los TF <1 significa que la planta fija los metales en la raíz cumpliendo un papel fitoestabilizador.

Observando los valores obtenidos de TrC y TF se podría decir que Brassica Juncea L. cultivada sobre este tipo de tratamientos tendría un papel marcadamente fitoestabilizador.

7.2 Efecto de la adición de compost y biochar y de la revegetación con Brassica juncea L. sobre el suelo de la balsa de decantación de la mina de Touro (capítulo 2-anexo II)

7.2.1 Evolución del pH

El efecto positivo sobre el pH, una vez aplicados al suelo de la balsa de decantación los tratamientos elaborados con compost o con compost combinado con biochar (con o sin planta), fue más notable en la profundidad 0-15 cm (Figura 1, anexo II). En esta profundidad, al final del tiempo experimental, la aplicación de compost combinada con biochar (SCB y SCBP) presentó valores significativamente mayores de pH en comparación con la aplicación del compost en solitario (SC y SCP) (Figura 1, anexo II). Observando el comportamiento de los tratamientos a lo largo del experimento, en las profundidades 15-30cm y 30-45cm, salvo alguna excepción puntual, todos los tratamientos consiguieron aumentar el pH, destacando el aumento de pH provocado por el tratamiento que combinaba compost con biochar y Brassica juncea L. (SCBP) (Figura 1, anexo II).

Como hemos comentado en el apartado anterior (7.1), el mayor aumento de pH de los tratamientos que contienen biochar demuestra la capacidad del biochar para mejorar el efecto de las enmiendas orgánicas, lo cual ya fue descrito anteriormente por Fowles (2007). El alta área superficial y la carga negativa del biochar son los factores que más influyen en el aumento de pH (Karer et al., 2015, Puga et al., 2015, Beesley y Marmiroli, 2011).

Este aumento de pH es importante y afecta positivamente en la reducción de las concentraciones fitodisponibles de metales (Puga et al., 2016). Esto se confirma con las correlaciones de Pearson existentes entre el pH y las concentraciones fitodisponibles de Cu, Pb y Ni, con valores de r=-0,93 para el Cu, r=-0,88 para el Pb, r=-0,90 para el Ni (p˂0,01). La única excepción fue el Zn con una r=0,60 y P˂0,01, probablemente debido a las altas concentraciones de Zn que presentaba el compost y que fueron aportadas al suelo con esta enmienda. Al aumentar

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Discusión general el pH también mejora la disponibilidad de nutrientes para las plantas (Hazelton y Murphy, 2007). Al realizar correlaciones de Pearson entre el pH y los nutrientes, los datos confirmaron lo propuesto por Hazelton y Murphy (2007), obteniéndose correlaciones positivas entre el pH y el Ca (r=0,76, P˂0,01), el K (r=0,61, P˂0,01), N (r=0,91, P˂0,01), el Na (r=0,72, P˂0,01), el Mg (r=0,83, P˂0,01).

El tratamiento que combinaba compost, biochar y vegetado con Brassica juncea L. fue el que provocó un mayor aumento de pH con respecto al resto de tratamientos. Este aumento de pH, en este caso, podría potenciar la mejora de la disponibilidad de nutrientes y la supervivencia de las plantas, en este caso Brassica juncea L., durante la etapa temprana del establecimiento de vegetación (Reverchon et al., 2015, Shrestha y Lal, 2011; Zhang et al., 2013).

7.2.2 Evolución del contenido total de carbon (CT)

La aplicación de compost y compost combinado con biochar (con y sin planta) provocó un claro aumento de CT en la profundidad 0-15cm en comparación con los controles (S y SS) (Figura 4, anexo II). La aplicación de compost y biochar fue la que provocó un mayor aumento de CT (Figura 4, anexo II). El aumento de CT en la profundidad 15-30cm se hizo notable a partir del segundo tiempo, posiblemente debido a que fue cuando éste empezó a migrar. Al final del experimento, el tratamiento que combina compost y biochar con Brassica juncea L. fue el que provocó un mayor aumento de CT. Por último, en la profundidad 30-45cm, la aplicación de compost y biochar aumento de forma signifivativa y pronunciada el contenido de CT. En el último tiempo el tratamiento SCBP, como ocurrió en la profundidad 15-30cm, fue el que presentó un contenido significativamente mayor de CT. Este mejor comportamiento de SCBP fue debido probablemente a la acción de las Brassica juncea L. cultivadas cuyas raíces facilitan la migración de carbono através del perfil. Si se observan los datos de CT se observa una clara tendencia ascente de CT en las tres profundidades desde el tiempo 1 al tiempo 3 en todos los tratamientos utilizados, esta tendecia se traduce en que cuanto más profundizamos, más tiempo se tarda en observar un cambio importante del contenido de CT.

El aumento de CT provocado por los tratamientos se debe, por un lado, a los elementos con los que se elaboró el compost y, por otro, al biochar ya que, como se puede observar en la Tabla 2 (anexo II), las enmiendas (compost y biochar) tienen un alto contenido en CT, si bien el TC del biochar es más del doble. Los tratamientos elaborados solo con compost aumentaron el CT debido a que los residuos con los que se elaboró tienen una alta carga orgánica como, por ejemplo, el estiércol de caballo (Pérez-Esteban et al., 2014), el estiércol de conejo (Islas-Váldez et al., 2016) o las algas (Stutter, 2015). Este aumento de CT provocado por el compost se vio potenciado una vez que se añadió biochar, como en el tratamiento SCBP. Esto se debe a que el

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Discusión general biochar ultilizado en este experimento presentó una concentración de CT de 676 g/kg. Biederman y Harpole (2013) y Madiba et al. (2016) ya demostraron que el biochar provoca un aumento de CT en el suelo. El aumento de materia orgánica, además de aumentar el contenido de CT, aumenta el contenido de nutrientes (Wijesekara et al., 2016); además el carbono aportado en los tratamientos elaborados con biochar el altamente recalcitrante (IBI, 2015).

-1 7.2.3 Evolución de la capacidad de intercambio catiónico (CEC) (cmol(+)kg ), saturación de bases (V%) y saturación de aluminio (Al%)

En la profundidad 0-15 cm, la CEC fue mayor cuando se combinaban compost y biochar con Brassica juncea L. tanto en el primer como en el segundo tiempo (Tabla 3, anexo II). Sin embargo, en el tercer tiempo, la CEC fue mayor en el tratamiento que combina compost y Brassica juncea L. Analizando los valores de saturación de bases (%V) y del aluminio de cambio (Al%), se observa como para todos los tratamientos %V y Al% presentron unos valores medios de 100% y 0% respectivamente (Tabla 3, anexo II). Queda, por tanto, patente la efectividad de los tratamientos a la hora de aumentar la CEC en comparación con los controles (S y SS). Este aumento de CEC es importante por que está relacionado con la fitodisponibilidad de metales, el pH, y la disponibilidad de nutrientes (Chandra et al., 2014; Hazelton y Murphy, 2007). En el primer y segundo tiempo el mayor efecto de los tratamientos que contenían biochar se debe posiblemente a la alta capacidad de retención de cationes por parte del biochar, inducida por su alta aromaticidad, área superficial específica y carga negativa (Karer et al., 2015, Puga et al., 2015).

En la siguiente profundidad (15-30cm) de nuevo SCBP fue el tratamiento que presentó los valores más altos de CEC. Sin embargo, en el tercer tiempo, en esta profundidad fue el suelo de la balsa de decantación sin tratar (S) el que presentó una CEC mayor (Tabla 3, anexo II). Calculando el %V y %Al, se observa cómo los tratamientos presentan, en general, una mayor %V y menor Al% en todos los tiempos en comparación con S (Tabla 3, anexo II). En el tercer tiempo destaca que, alcanzando S el valor más alto de CEC, fue SCBP el que presentó una mayor %V y menor %Al (%V de 96%, %Al de 3,56) (Tabla 3, anexo II). Los datos de %V y %Al de los tratamientos, tanto con biochar como sin él, nos indican que los contenidos en cationes básicos en los tratamientos siempre son más altos que en en el suelo de la balsa de decantación sin tratar. En la última profundidad (30-45cm), en los dos primeros tiempos, el efecto de los tratamientos fue el mismo que en la profundidad 15-30 cm aunque en el tercer tiempo el efecto es más difuso (Tabla 3, anexo II).

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Discusión general

7.2.4 Evolución del contenido total de nitrógeno (NT) en la profundidad 0-15 cm

El contenido de nitrógeno total (NT) en el suelo de la balsa de decantación y en el suelo de la balsa de decantación tratado con arena no fue detectable (límite de detección NT <0,1%) a lo largo del tiempo experimental en las tres profundidades estudiadas (Figura 5, anexo II). Una vez aplicado el compost y el compost con biochar (con y sin planta) el aumento de NT fue significativo en la profundidad 0-15 cm (Figura 5, anexo II). Sin embargo, este aumento de nitrógeno no se produjo en la segunda y tercera profundidad a lo largo del tiempo (Figura 5, anexo II). Esto es probablemente debido a que el nitrógeno se acumuló en la profundidad 0-15 cm al ser la capa más superficial y donde más efecto ejercen los tratamientos aplicados. Por otro lado, en las profundidades 15-30 cm y 30-45 cm posiblemente el nitrógeno se perdió rápidamente por migración al ser formas poco estables. La mayoría de los residuos biológicos contienen nitrógeno en forma orgánica en lugar de en las formas inorgánicas, que están fácilmente disponibles para las plantas, pero también tienen la tendencia a lixiviarse (Wijesekara et al., 2016).

Como hemos comentado, en la profundiad 0-15 cm los tratamientos consiguieron aumentar el NThasta un valor medio de 20 mg/kg. Incrementar el NT es muy importante a la hora de recuperar un suelo como el de la balsa de decantación, el cual presentó un déficit muy alto de TN, ya que la disponibilidad de N en las plantas es un aspecto crucial en de la calidad del suelo y, a menudo, el nitrógeno representa una limitación inmediata en el desarrollo vegetal (Nguyen et al., 2017)

Los valores más altos de TN a lo largo del experimento los presentó el suelo de la balsa tratado solo con compost y Brassica juncea L. (Figura 5, anexo II). Este mayor contenido deNT en SCP se debió posiblemente a que este tratamiento se elaboró con un 15% de compost (21,3 mg/kg de NT), mientras que los tratamientos que combinan compost y biochar se elaboraron con un 11% de compost y 4% de biochar, lo que significa que contienen una menor proporción de compost e incluyen biochar, el cual presentaba una media de 5,34mg/kg de NT. Por lo tanto, debido a esta clara diferencia de contenido de NT entre el compost y el biochar, este último tendría un efecto diluidor. A su vez, el hecho de que el tratamiento SCP (compost+Brassica Juncea L.) presentase un mayor contenido de NT en comparación con el tratamiento elaborado solo con compost (SC) se puede deber al efecto de las Brassica juncea L. sobre la rizósfera. Los exudados radiculares son factores importantes que estructuran la comunidad bacteriana de la rizósfera. Estas comunidades bacterianas no simbiontes son fijadoras de nitrógeno, y suelen asociarse con ciertas especies de brassicas (Germida et al., 1998, Misko y Germida, 2002). Sin embargo, este comportamiento no se repitió entre los tratamientos elaborados con compost y biochar vegetados y sin vegetar, en este caso SCBP al final de los 11 meses presentó un contenido menor de NT, lo cual se puede achacar a que el biochar favorece la disponibilidad de nutrientes para las plantas.

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Existen estudios que demuestran un mejor crecimiento de la vegetación cuando se aplica biochar y una mayor eficiencia en el uso de N y P por parte de la vegetación (Zhu et al., 2014).

7.2.5 Relación carbono y nitrógeno (C/N) en la profundidad 15 cm

La relación entre el carbono y nitrógeno (C/N) una vez se aplicó tanto el compost como el compost combinado con biochar (con o sin planta) aumentó en general hasta el rango óptimo propuesto por Troeh y Thompson (2005) (Figura 2), rango que abarca desde C/N ˃10 a C/N ˂30. Sólo se presentó una excepción a los tres meses, donde la aplicación de compost vegetado con Brassica juncea L. no alcanzó el valor mínimo de C/N.

Figura 2. Relación del contenido de carbon y nitrógeno (C/N) en la profundidad 0-15cm.

Como se comentó en el apartado anterior 4.1.5 los valores de C/N dentro del rango propuesto por Troeh y Thompson (2005) indican que el nitrógeno está en forma mineralizada, lo que es importante para las plantas ya que es en el estado que pueden absorber el nitrógeno.

Los tratamientos que combinaban compost y biochar (con o sin planta) presentaron una C/N mayor que la aplicación directa de compost (con o sin planta), la causa de esta diferencia en los valores de C/N se debe a que los tratamientos elaborados con biochar presentaron un contenido en cabono mayor (Figura 4, anexo II).

7.2.6 Factores limitantes para la producción vegetal en la profundidad 0-15 cm

Los factores limitantes para la producción y desarrollo vegetal se analizaron en la profundidad 0-15 cm (Tabla 5, anexo II). Como ya fue comentado en el capítulo 7.1, el suelo de la balsa de decantación está afectado por varios de estos factores (Factor c, Factor Ca, Factor e, Factor K, Factor n, Factor N) los cuales detallaremos a continuación.

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El suelo de la balsa de decantación está afectado por el Factor c a lo largo de todo el experimento debido a su bajo pH (Tabla 5, anexo II). Ninguno de los tratamientos está afectado por este factor, lo cual se debe a que los tratamientos aumentan el pH, factor muy importante a la hora de reducir las concentraciones fitodisponibles de metales y mejorar la disponibilidad de nutrientes.

El Factor Ca afectó al suelo de la balsa de decantación durante todo el experimento. Ninguno de los tratamientos se vio afectado por este factor (Tabla 5, anexo II). Los altos valores de este factor presentados por los tratamientos se deben al Ca aportado por los elementos con los que se elaboró el compost. El compost aportó 6455±153 mg/kg de Ca frente a 13,3±0,02 mg/kg del suelo de la balsa de decantación, y por otro lado el compost presentó un valor de 32,2±0,76

-1 -1 cmol(+)kg frente a un valor de 0,07 cmol(+)kg de S.

El suelo de la balsa de decantación está afectado por el Factor e en el primer y segundo tiempo, dejando de estarlo en el tercer tiempo (Tabla 5, anexo II). Hemos de recordar que, aunque en S la CEC aumentó en el tercer tiempo, fue debido al aumento del contenido de Al+3 (Al% de 61,9). En lo que respecta a los tratamientos, ninguno se vio afectado por el Factor e. Que S deje de estár afectado por el Factor e al aplicar los tratamientos es de gran importancia, ya que la CEC está relacionada con la biodisponibilidad de metales, pH y nutrientes (Chandra et al., 2014; Hazelton y Murphy, 2007).

En el suelo de la balsa de decantación, los valores de K y N fueron críticos, estando S afectado por los Factor K y Factor N durante todo el experimento (Tabla 5, anexo II). Todos los tratamientos aplicados demostraron su efectividad a la hora de aportar K y N ya que ninguno de los tratamientos se vio afectado por dichos factores. El aumento de N es importante para mejorar la calidad de suelos degradados y a la hora de intentar establecer una cobertura vegetal como ya propusieron Christensen (2004) o Zanuzzi et al. (2013). Además, tanto el suelo de la balsa de decantación sin tratar como tratado se vio afectado por el Factor n como ocurría en el tratamiento con tecnosol (capítulo 7.1).

7.2.7 Evolución de la biomasa cosechada de las Brassica juncea L. cultivadas

Uno de los problemas más importantes en los suelos de mina es que presentan una serie de problemas que impiden a la vegetación instaurarse de forma estable (Wong, 2003). Esto quedó patente en este experimento ya que las Brassica juncea L. no crecieron en el suelo de la balsa de decantación (S) (Figura 6, anexo II). Recordemos que el suelo de la balsa de decantación se vio afectado por distintos factores para la producción vegetal como son el Factor c, Factor Ca, Factor e, Factor K, Factor n, Factor N (Tabla 5, anexo II). Sin embargo, las Brassica juncea L. se desarrollaron sin problema una vez se aplicó al suelo de la balsa de decantación compost (SCP)

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Discusión general o la combinación de compost y biochar (SCBP) (Figura 6, anexo II). Basándonos en los datos de altura de las Brassica juncea L., éstas tuvieron un mejor desarrollo en el tratamiento SCP, pero al analizar los datos de biomasa, ésta fue siempre significativamente mayor en las Brassica juncea L. cultivadas en el tratamiento SCBP, de hecho, esta biomasa fue aumentando a lo largo del experimento (Figura 6, anexo II).

El tratamiento del suelo de la balsa de decantación con compost y biochar es el que menor concentración de NT presentó al final del experimento. Sin embargo, las Brassica juncea L. cultivadas sobre él, presentaron mayor biomasa. Este mejor desarrollo de las Brassica juncea L. en el tratamiento SCBP se debe a características del biochar como la mejora de la disponibilidad de nutrientes para las plantas como ya ha sido demostrado por Zhu et al. (2014). Quizás esta mejor disponibilidad de nutrientes para las plantas una vez aplicado biochar al suelo es la que provoca el menor contenido de NT en el suelo tratado con compost y biochar frente al tratado solo con compost. Autores como Gwenzi et al. (2015) demostraron que el biochar es capaz de reducir la migración de nitratos, aumentar el uso eficiente de los nutrientes por las plantas, aumentar el pH, estimular la actividad microbiana del suelo y mejorar la estructura del suelo. Con respecto a estos beneficios del biochar también otros autores como Anawar et al. (2015) realizaron una comparación entre la siembra de semillas sin biochar frente a la adición de biochar a la hora de sembrar. Los autores en este trabajo concluyeron que la adición de biochar a un suelo influye positivamente en la germinación de las semillas, el crecimiento de las plantas y la cubierta vegetal.

7.2.8 Evolución de las concentraciones fitodisponibles de Cu, Pb, Ni, Zn

El efecto de los tratamientos aplicados sobre el suelo de la balsa de decantación en la profundidad 0-15 cm fue claro a la hora de reducir las concentraciones fitodisponibles de Cu, Pb, Ni y Zn salvo una excepción, el Zn (Tabla 3, capítulo 2). Esta excepción se produjo cuando se aplicó el compost sin combinar con biochar. En los casos que se aplicó solo compost, las concentraciones fitodisponibles de Zn fueron significativamente superiores en comparación con los controles y con la combinación de compost con biochar (con o sin planta) (Tabla 3, capítulo 2). Con esto queda patente el efecto del biochar a la hora de potenciar la capacidad del compost en la reducción de las concentraciones fitodisponibles de los metales estudiados.

Analizando el porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn al final del tiempo experimental en esta profundidad (0-15 cm) (Tabla 2), se observó como todos los tratamientos presentaron porcentajes más bajos que los controles S y SS. Además, comparando los porcentajes de los diferentes tratamientos se observó como los valores más bajos predominan en los tratamientos SCB y SCBP.

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Tabla 2. Profundidad 0-15 cm, y tiempo 3. Porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn.

% S SS SC SCP SCB SCBP

Cu 7,77 7,41 0,16 0,24 0,15 0.23

Pb 1,55 1,16 0,31 0,23 0,36 0.41

Ni 32,5 0,38 0,30 2,86 0,07 0,25

Zn 6,81 1,60 4,07 5,05 2,75 3,17

La disminución de las concentraciones fitodisponibles de Cu, Pb y Ni por parte de los tratamientos elaborados sólo con compost (SC y SCP) se debe a que los residuos con los que se elaboró dicho compost tienen un alto contenido en materia orgánica y altas concentraciónces de cationes básicos, lo que repercute directamente en la CEC y el pH (Islas-Váldez et al., 2016, Pérez-Esteban et al., 2014, Stutter, 2015). Además, este compost tiene entre sus componentes algas marinas. A algunas especies de algas se les atribuye una alta capacidad de biosorción de metales cuando son empleadas como enmiendas, provocando una reducción en las concentraciones fitodisponibles de metales. Esto se debe a la pared celular de las algas, compuesta por un esqueleto fibrilar y una matriz de inclusión amorfa, que aumentan la atracción electroestática y la capacidad de complejación (Figueira et al., 2000). Como se comentó anteriormente, los tratamientos que combinaban compost y biochar redujeron las concentraciones fitodisponibles de los metales estudiados, debido a que el biochar provocha una mejora en los efectos positivos de las enmiendas elaboradas con residuos aumentando el CT, CEC y pH (Chandra et al., 2014, Fowles, 2007, Puga et al., 2016).

La influencia de los tratamientos sobre el suelo de la balsa de decantación no fue tan claro en la reducción de las concentraciones fitodisponibles de metales en la profundidad 15-30 cm (Tabla 4, capítulo 2). El tratamiento SCBP fue el que presentó un mejor comportamiento a la hora de reducir las concentraciones fitodisponibles de los metales estudiados. Este tratamiento, a lo largo de todo el tiempo experimental, mostró una tendencia a reducir las concentraciones fitodisponibles de Cu y Pb, incluso la de Zn en el segundo tiempo (Tabla 4, capítulo 2). Realizando una comparación entre el porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn al final de los 11 meses (Tabla 3), se obtuvo que el compost solo o combinado con biochar (con o sin planta) en comparación con S presentó en general porcentajes más bajos de Pb, Ni y Zn fitodisponible con respecto al total, pero no de Cu. El Cu fue el metal más problemático al final del tercer tiempo posiblemente debido, por un lado, a las altas concentraciones fitodisponibles del material de partida y, por otro, a que el compost presentaba

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Discusión general una concentración pseudototal de Cu de 193 mg/kg que, en esta profundidad, al disminuir el pH, puede provocar que este metal aumente su concentración fitodisponible.

Tabla 3. Porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn. Profundidad 2, tiempo 3.

% S SS SC SCP SCB SCBP

Cu 10,9 13,1 12,5 15,0 18,2 20,6

Pb 1,28 1,17 1,29 1,18 1,27 1,11

Ni 29,6 18,9 23,3 16,3 20,5 24,7

Zn 6,82 5,41 5,92 5,70 4,98 9,67

En la profundidad 30-45cm, al final del tiempo experimental, el tratamiento más eficaz fue el que combina compost con biochar y Brassica juncea L (SCBP), el cual presentó concentraciones fitodisponibles de Cu Pb, Ni y Zn menores en comparación con el suelo de la balsa de decantación y también, en general, con el compost aplicado en solitario o combinado con biochar sin vegetar. Cabe destacar la existencia de diferencias significativas en las concentraciones fitodisponibles de Cu, Pb y Zn obtenidas entre la aplicación de compost combinado con biochar y Brassica juncea L. y el compost combinado solamente con biochar (Tabla 5, capítulo 2). Los tratamientos presentaron un buen comportamiento si comparamos el porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn al final de los 11 meses (Tabla 4).

Tabla 4. Porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn. Profundidad 3, tiempo 3.

% S SS SC SCP SCB SCBP

Cu 11,4 12,4 11,1 11,3 14,8 11.5

Pb 1,35 1,50 1,15 1,18 1,46 0,97

Ni 28,8 22,1 24,8 19,6 18,3 19,7

Zn 9,56 6,05 7,45 7,11 5,40 5,85

Hay algunas excepciones, como en el caso de Cu, en los tratamientos SCB y SCBP, pero en esta profundidad (30-45 cm) predomina el suelo de la balsa de decantación el cual presentó

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Discusión general altas concentraciones de Cu debido a su origen. La otra excepción fue el Pb en el tratamiento SCB. Por ello, como solo se observaron excepciones puntuales, decimos que los tratamientos tienen un buen comportamiento si comparamos porcentaje de la concentración fitodisponible frente a la concentración pseudototal (Tabla 4).

7.2.9 Evolución del Coeficiente de tranferencia (TrC) y Factor de translocación (TF) en las Brassica juncea L. cultivadas sobre SCP y SCBP

En apartados anteriores ya ha sido detallado cómo para que una planta se considere hiperacumuladora debe tener un valor del Factor de Translocación (TF) mayor de 1 (Baker y Brooks, 1989) o poseer un valor del Coeficiente de Transferencia (TrC) también superior a 1 (Busuioc et al., 2011). Las Brassica juncea L. cultivadas en el suelo de la balsa de decantación tratado con compost y compost combinado con biochar no presentaron valores superiores a 1 de ninguno de estos dos factores (TF y TrC, Figuras 5 y 6, capítulo 2) para ninguno de los tiempos y metales estudiados (Cu, Pb, Ni, Zn). Las Brassicas juncea L. cultivadas en estos tratamientos no presentaron capacidad fitoextractora. Esto no quiere decir que no tengan un papel importante a la hora de fitorremediar, sino que, en vez de jugar un papel fitoextractor lo harían con un papel fitoestabilizador. Esta función fitoestabilizadora ha sido demostrada por diversos autores, basándose en valores de TrC y TF, por ejemplo, Kidd et al. (2009) concluyeron que las plantas efectivas a la hora de fitoestabilizar tienen coeficientes de transferencia raíz-tallo bajos, y Nouri et al. (2009) que si los valores de TF son menores a 1 significa que la planta en vez de translocar estos metales desde la raíz hasta el brote, los fija en la raíz cumpliendo un papel fitoestabilizador.

7.3 Comparativa del efecto de tratamientos elaborados con compost y biochar frente a la combinación de tecnosol y biochar, ambos vegetados con Brassica juncea L. (anexos III y IV)

En este apartado compararemos las diferencias en los efectos que provocan los tratamientos SCBP (compost+biochar+Brassica juncea L.) y STBP (tecnosol+biochar+ Brassica juncea L.) al aplicarlos sobre el suelo de la balsa de decantación (S).

7.3.1. Evolución del pH

El aumento de pH en el suelo de la balsa de decantación fue significativo (P < 0.05) después de la aplicación tanto de compost combinado biochar como la combinación de tecnosol y biochar, sobre todo en las profundidades 0-15 cm y 15-30 cm. En la profundidad 30-45cm,

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Discusión general aunque también aumentó significativamente lo hizo de forma menos pronunciada (Tabla 3, anexo IV).

En la primera profundidad (0-15 cm), al final del experimento, el pH aumentó de 2,63 en S a 7,53 y 7,48 en SCBP y STBP respectivamente, no mostrándose diferencias significativas entre estos dos valores de pH. Esto puede ser debido a que los valores de pH fueron significativamente distintos entre el compost y el tecnosol, pero con valores similares (Tabla 2, anexo IV) y el porcenjate de biochar fue el mismo en ambos tratamientos (4% de biochar, pH 9.90). Diferentes autores como Karer et al. (2015) y Zhang et al. (2013) describieron la influencia del biochar sobre el aumento del pH del suelo. Además, como se comentó anteriormente, este aumento de pH mejora la disponibilidad de nutrientes para las plantas (Hazelton y Murphy, 2007) y afecta positivamente en la reducción de las concentraciones fitodisponibles de metales (Puga et al., 2016). Al final del tiempo experimental, en la segunda y tercera profundidad, la aplicación de compost y biochar provocó un aumento de pH mayor en comparación con la aplicación de tecnosol y biochar. Este mayor aumento pudo ser debido a que el compost presentó un pH ligeramente superior al tecnosol (Tabla 2, anexo IV). Autores como Rodríguez-Vila. (2015) ya demostraron que la combinación de compost y biochar aumenta más el pH que la combinación de tecnosol y biochar.

7.3.2 Evolución del contenido total de carbon (CT)

Como se detalló en los capítulos anteriores 7.1 y 7.2 el contenido del CT en el suelo de la balsa de decantación fue muy bajo a lo largo de todo el experimento en las tres profundidades estudiadas. Una vez se aplicaron sobre S los tratamientos SCBP (compost+biochar+Brassica juncea L.) y STBP (tecnosol+biochar+Brassica juncea L.), el contenido de TC aumentó significativamente (Figura 3, anexo IV). Al final del tercer tiempo, en la profundidad 0-15 cm, aunque el compost presentó un mayor contenido de CT en comparación con el tecnosol (Tabla 2, anexo IV), fue la combinación de tecnosol y biochar la que presentó un contenido significativamente mayor de CT (Figura 3, anexo IV). Esto nos indica que el carbono aportado por el tecnosol posiblemente sea más estable, ya que el carbono aportado por el biochar fue el mismo en los dos tratamientos. Tanto en la profundidad 15-30 cm como en la profundidad 30-45 cm, fue la aplicación de compost y biochar la que provocó mayores contenidos de CT en el suelo de la balsa, posiblemente porque el carbono del compos tsufra una perdida por migración de la primera profundidad a las dos siguientes. El problema de la pérdida de eficiencia del compost a largo plazo ya fue demostrado por autores como Walker et al. (2004).

El aporte de carbono por parte de ambos tratamientos al suelo de la balsa de decantación es muy importante ya que, al aumentar la concentración de carbono orgánico, aumenta la

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Discusión general posibilidad de retener agua y nutrientes en forma biodisponible. También afecta positivamente a la hora de reducir la cantidad de metales biodisponibles, mejora la estructura del suelo y otras propiedades físicas (Karer et al., 2015; Lal, 2006, Puga et al., 2015, Rodríguez-Vila et al., 2015). Por otra parte, el tipo de carbono del biochar es altamente recalcitrante, lo que significa que no se mineraliza rápidamente y se almacena en el suelo durante mucho tiempo (IBI, 2016).

-1 7.3.3 Evolución de la capacidad de intercambio catiónico (CEC) (cmol(+)kg )

La aplicación tanto de compost con biochar como de tecnosol combinado con biochar aumentaron de forma significativa la CEC en S a lo largo del experimento y en las profundidades estudiadas, salvo la excepción del último tiempo en la profundidad 30-45 cm (Tabla 3, anexo IV). Este aumento de la CEC provocado por los tratamientos en el suelo de la balsa de decantación se debe posiblemente al aumento en materia orgánica, ya que un aumento o descenso del contenido de materia orgánica en el suelo influye en la CEC (Agegnehu et al., 2015). Las correlaciones de Pearson realizadas entre CT y CEC confirman lo anterior ya que obtenemos una correlación positiva entre estos dos factores (r=0,92; P˂0,01)

Al final de los 11 meses de experimento, en la profundidad 0-15 cm, no se observaron diferencias significativas en la CEC entre el tratamiento SCBP y el STBP pese a lo que se podría esperar, ya que la CEC del tecnosol era mayor que la del compost (Tabla 3, anexo IV). La ausencia de diferencias significativas entre la CEC del tratamiento SCBP y la del STBP al final del tiempo experimental se puede deber a la adición del biochar al elaborar ambos tratamientos. El biochar, al ser un material con una estructura porosa y amplia superficie específica, podría, con el paso del tiempo, igualar y estabilizar la CEC en los tratamientos. En el tratamiento SCBP y en el STBP la saturación de bases (%V) fue del 100% y del 99% para SCBP y STBP respetivamente, lo que nos indica el alto contenido de cationes básicos. El aumento de CEC mediante el aumento de cationes básicos es importante ya que repercute en el aumento de pH, de hecho, la CEC está correlacionada con el pH con una correlación de Pearson positiva con una r=0,88 y P˂0,01.

En la profundidad 15-30 cm, la CEC siempre fue significativamente superior en el tratamiento SCBP (Tabla 3, anexo IV), posiblemente debido a que sus componentes migran con

-1 mayor facilidad por el perfil. Sin embargo, en SCBP la CEC pasa de 10,4 cmol(+)kg en el primer

-1 - tiempo a 13,6 cmol(+)kg en el último, en cambio en el tratamiento STBP pasa de 2,85 cmol(+)kg

1 -1 en el primer tiempo a 10,0 cmol(+)kg en el tercero, es decir, el aumento dentro del tratamiento es mayor en STBP (Tabla 3, anexo IV ). Desglosando la CEC en %V y Al% en el último tiempo la %V en SCBP fue mucho mayor que en STBP (Tabla5). En el tratamiento SCBP, en el tercer tiempo, la %V fue de 96,4% valor cercano al 100% presentado en la primera altura, lo que nos indica la fuerte influencia de este tratamiento a esta profundidad (Tabla 5)

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Tabla 5. Evolución de la saturación de bases (V%) y la saturación de aluminio (Al%) en las tres profundidades y durante los 11 meses de experimento

S SS SCBP STBP

V% Tiempo 1 39.4 43.7 100 99.9

Al% 60.5 56.2 0 0.01

15 15 cm

- V% Tiempo 2 36.1 52.9 100 99.9

Al% 63.8 47.0 0 0.01

Profundidad 0 V% Tiempo 3 41.8 44.2 100 99.9

Al% 58.1 55.7 0 0.01

V% Tiempo 1 36.3 27.8 48.9 62.8

Al% 63.6 72.1 51.1 37.2

30 30 cm - V% Tiempo 2 37.5 47.6 44.1 47.9 Al% 62.2 51.6 55.9 52.1

Profundidad 15 Tiempo 3 38.3 39.0 96.4 71.4 Al% 61.6 60.9 3.56 28.6

V% Tiempo 1 41.1 26.8 35.0 34.7

Al% 58.8 73.11 65.0 65.3

45 45 cm

30 Tiempo 2 V% 32.8 59.5 42.1 35.5

Al% 67.1 40.5 57.9 64.5

Profundidad Tiempo 3 V% 40.2 34.0 43.5 38.5 Al% 59.8 66.0 56.5 61.5

En la profundidad 30-45 cm, la aplicación al suelo de la balsa de decantación de compost combinado con biochar y tecnosol con biochar no consiguió mantener el aumento de CEC en comparación con S (Tabla 3, anexo IV). En los dos primeros tiempos, ambos tratamientos mantuvieron la tendencia a aumentar la CEC en comparación con el suelo de la balsa de decantación. Sin embargo, en el tercer tiempo, la CEC de S es significativamente mayor. Dentro de los tratamientos (SCBP y STBP) de nuevo el tratamiento que combinaba compost con biochar y Brassica juncea L. fue el que presentó una mayor CEC. La saturación de bases presentada por el tratamiento SCBP fue mayor en comparación con la del suelo de la balsa de decantación sin tratar y con la del tratamiento STBP al final del tiempo experimental (Tabla 5).

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7.3.4 Evolución del contenido total de nitrógeno (NT) en la profundidad 0-15 cm.

Como se comentó en los capítulos 7.1 y 7.2 de la presente Tésis, el contenido en nitrógeno en el suelo de la balsa de decantación y de los tratamientos STBP y SCBP solo fue detectable en la profundidad 0-15 cm. Una vez aplicados ambos tratamientos, el aumento de NT fue significativo durante todo el tiempo experimental (Tabla 4, anexo IV). La combinación de compost con biochar y vegetado con Brassica juncea L. presentó un contenido superior al tratamiento elaborado con tecnosol y biochar también vegetado con Brassica juncea L. Este mayor contenido de NT se debe posiblemente a que la media de NT en el compost fue de 21,3 mg/kg mientras que el tecnosol fuede 17,6 mg/kg, siendo en el biochar mucho menor 5,34 mg/kg, por lo que el contenido de nitrógeno en los tratamientos lo determinó la influencia de compost y tecnosol. Observando los datos de NT a lo largo de todo el experimento cabe destacar que son valores muy estables, sobre todo en el caso del tratamiento STBP. Esta estabilidad se debe a la capacidad del biochar de fijar nitrógeno. Muchos investigadores informaron de la mayor retención de N en el suelo y la reducción de la emisión de óxido nitroso y migración de nitratos después de la adición de biochar (Laird et al., 2010, Steiner et al., 2008, Yao et al., 2012). En capítulos anteriores, en los apartados referentes a la evolución del nitrógeno, ya se comentó que este aumento del mismo es muy importante al ser un nutriente esencial y un factor limitante para el desarrollo de la vegetación (Christensen, 2004).

7.3.5 Relación carbono y nitrógeno (C/N) en la profundidad 15 cm

La relación entre el carbono y el nitrógeno (C/N), como se comentó en los apartados 7.1.5 y 7.2.5, aumentó al aplicar los tratamientos en el suelo de la balsa de decantación. Tanto la aplicación de compost combinado con biochar y vegetado con Brassica juncea L. como la aplicación de tecnosol mezclado con biochar y vegetado con Brassica juncea L. presentaron valores de C/N dentro del rango óptimo que abarca entre una C/N ˃10 a una C/N ˂30 (Troeh y Thompson, 2005) (Figura 3). La mejor C/N la presentó el tratamiento que combinaba tecnosol y biochar con Brassica juncea L., esto pudo ser debido a que este tratamiento presentó un mayor contenido en carbono (Figura 3, capítulo 6) y un menor contenido en nitrógeno (Figura 4, capítulo 6) en comparación con el tratamiento elaborado con compost combinado con biochar y Brassica juncea L.

Este aumento de C/N es beneficioso para las plantas, ya que indica que el nitrógeno está mineralizado (Troeh y Thompson, 2005) y que puede ser absorbido por las plantas. Recordemos que el nitrógeno es un factor limitante para el desarrollo vegetal, el cual afectaba al suelo de la balsa de decantación. Como se observó en la figura 5 del capítulo 6 las Brassica juncea L. no

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Figura 3. Relación del contenido de carbon y nitrógeno (C/N) en la profundidad 0-15cm.

7.3.6 Evolución del contenido en nutrientes

Al observar la evolución del contenido de nutrientes en el suelo de la balsa de decantación una vez aplicados el tecnosol y compost combinados con biochar, queda patente la eficacia de ambos tratamientos para aumentar los contenidos de Ca, K, Mg y Na (Tabla 4, anexo IV). Este aumento fue más pronunciado a lo largo del tiempo en las profundidades 0-15cm y 15-30 cm. En cuanto a la profundidad 30-45, el efecto solo fue claro en el primer y segundo tiempo.

En la profundidad 0-15 cm, la aplicación de compost y biochar aumentó de forma significativa el contenido de los nutrientes en comparación con la aplicación de tecnosol y biochar en el primer tiempo, pero al ir transcurriendo el experimento, el efecto de SCBP y STBP fue similar (Tabla 4, anexo IV). Sin embargo, analizando los valores de los nutrientes en cada tratamiento, se observó como, en SCBP, estos valores tienen una tendencia a descender mientras que, en STBP, la tendencia es a aumentar. Con estos datos se comprueba una pérdida de efecto del tratamiento elaborado con compost aunque se combine con biochar, algo que como se comentó anteriormente ya fue propuesto por Walker et al., (2004).

La pérdida de nutrientes en la profundiad 0-15 cm por parte del tratamiento SCBP con el transcurso del tiempo puede ser el motivo por el que dichos nutrientes aumenten en la profundidad 15-30 cm (Tabla 4, anexo IV). En la profundidad 15-30 cm, en el tercer tiempo, el tratamiento SCBP pesentó un contenido de nutrientes superior al suelo de la balsa de decantación y al tratamiento STBP, con la excepción de Mg.

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En la profundidad 30-45 cm, el efecto positivo de los tratamientos fue mayor en los dos primeros tiempos, con un aporte de nutrientes al suelo de la balsa de decantación similar (Tabla 4, anexo IV). En el tercer tiempo, los tratamientos van perdiendo efecto en esta profundidad, pero hemos de tener en cuenta que han transcurrido 11 meses, que los tratamientos mantuvieron durante este tiempo un cultivo permanente de Brassica juncea L., que no hubo re-tratamiento y que estamos hablando de una profundidad de 30-45 cm en un suelo de una balsa de decantación de una mina de cobre. En cuanto a la comparativa entre tratamientos, SCBP presentó valores superiores de Ca y Mg en comparación cona STBP en esta última profundidad en el tercer tiempo, en cuanto a los contenidos de K y Na no hubo diferencias significativas (Tabla 4, anexo IV). Esta eficacia a la hora de combinar compost y biochar a la hora de mejorar los nutrientes del suelo concuerda con los demostrado por Lui et al. (2012).

7.3.7 Evolución de la biomasa cosechada de las Brassica juncea L. cultivadas

La biomasa cosechada en las Brassica juncea L. cultivadas fue mayor en el tratamiento elaborado con compost y biochar en comparación con el tratamiento elaborado con tecnosol y biochar a lo largo de los 11 meses que duró el experimento (Figura 5, anexo IV). Esto se debe posiblemente a algunos de los parámetros estudiados anteriormente, por ejemplo, el tratamiento SCBP presentó un mayor contenido en TC, TN, una mayor CEC y además el aumento de nutrientes en líneas generales fue mayor en SCBP que en STBP. Además, el porcentaje de la concentración fitodisponible frente al porcentaje de concentración pseudototal de Cu, Pb, Ni y Zn al final de los 11 meses en la profundidad 0-15 cm como en la 30- 45cm y en algún caso en la 15- 30 cm fue menor en SCBP. Por todo ello el acondicionamiento realizado por este tratamiento mejoró las condiciones del suelo para que las Brassica juncea L. se desarrollasen mejor. Esto coincide con lo demostrado en varios estudios donde se ha indicado que la aplicación simultánea de biochar y compost podría conducir a una mayor fertilidad del suelo y un mejor crecimiento de las plantas (Fischer y Glaser, 2012; Schulz y Glaser, 2012).

7.3.8 Evolución de las concentraciones fitodisponibles de Cu, Pb, Ni, Zn

El efecto del tratamiento del suelo de la balsa con compost o tecnosol con biochar en la evolución de las concentraciones fitodisponibles de Cu, Pb, Ni y Zn al final de los 11 meses de experimento fue más claro en las profundidades 0-15 cm y 30-45 cm (Tablas 3-5, anexo III).

En la profundidad 0-15 cm, las concentraciones fitodisponibles de los metales estudiados en el suelo de la balsa de decantación se vieron reducidas una vez aplicados ambos tratamientos, excepto en el caso del Zn (Tabla 3, anexo III). Esta excepción se debe a las altas concentraciones fitodisponibles de Zn en el tecnosol ya que los elementos con los que se elaboró, como los lodos

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Discusión general de depuradora (Alvarenga et al., 2016) y restos de industrias agroalimentarias (González- González et al., 2013), presentan altas concentraciones fitodisponibles de Zn. En el caso del compost, los residuos con los que se elaboró como el estiércol de caballo (Pérez-Esteban et al., 2014), estiércol de conejo (Canet et al., 2008) y algas (Davis et al., 2003) suelen presentar concentraciones considerablesde Zn. El efecto positivo a la hora de reducir las concentraciones fitodisponibles de los metales estudiados, salvo la excepción comentada, se debe a que los tratamientos SCBP y STBP provocan un aumento de los valores de pH, CT y CEC. Estos factores, como demostraron autores como Chandra et al. (2014), Karer et al. (2015) ó Puga et al. (2016), están relacionados con la reducción de las concentraciones fitodisponibles de metales.

En esta primera profundidad, el tratamiento que mejor resultado presentó fue el tratamiento SCBP, el cual redujo significativamente las concentraciones fitodisponibles de Cu, Pb, Ni y Zn en comparación con el tratamiento STBP (Tabla 3, anexo III). Además, si observamos los datos del porcentaje de la concentración fitodisponible frente a la concentración pseudototal, el tratamiento SCBP presentó un porcentaje de Zn de 3,17% mientras que S y STBP presentaron valores de 6,81% y 31,7% respectivamente. Esto nos indica que el tratamiento SCBP es efectivo reduciendo las concentraciones fitodisponibles de todos los metales estudiados (Tabla 6).

Tabla 6. Profundidad 1, tiempo 3. Porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn

% S STBP SCBP

Cu 7,77 0,46 0,23

Pb 1,54 0,45 0,41

Ni 32,5 2,57 0,25

Zn 6,80 31,7 3,17

En la siguiente profundidad estudiada, 15-30 cm, los tratamientos SCBP y STBP presentaron concentraciones fitodisponibles de Cu inferiores a las del suelo de la balsa de decantación (Tabla 4, anexo III). Sin embargo, el comportamiento para los otros metales estudiados no fue tan claro como en la primera profundidad, ya que variaron a lo largo de los 11 meses. Sin embargo, el tratamiento SCBP presentó, al final del tiempo experimental, concentraciones fitodisponibles de Cu, Pb y Zn inferiores a las del tratamiento STBP (Tabla 4, anexo III).

Analizando en esta profundidad el porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn al final del tiempo experimental (Tabla 7), se

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Discusión general observa que las reducciones de estos porcentajes no predominan en un tratamiento por encima del otro. Sin embargo, si se realiza una comparación entre los diferentes porcentajes de los tratamientos y el suelo de la balsa de decantación, el porcentaje de la concentación fitodisponible frente a la concentración pseudototal fue menor en el tratamiento STBP, con la excepción del Zn, cuyo porcentaje en este caso fue practicamente igual en STBP y S (Tabla 7). Estos pocentajes fueron en general mayores en SCBP en comparación con STBP en esta profundidad 15-30 cm, posiblemente porque al descender en profundidad y transcurrir el tiempo, el compost perdió efecto coincidiendo con lo concluído por Walker et al. (2004). Esto puede ser debido a que los compuestos del tratamiento SCBP migren rápidamente por esta segunda profundidad sin quedar retenidos.

Tabla 7. Profundidad 2, tiempo 3. Porcentaje de la concentración fitodisponible a la concentración pseudototal de Cu, Pb, Ni y Zn

% S STBP SCBP

Cu 10,9 9,81 20,6

Pb 1,28 1,21 1,11

Ni 29,6 13,9 24,7

Zn 6,82 6,83 9,67

Al final de los 11 meses de experimento, en la profundidad 30-45 cm, ambos tratamientos STBP y SCBP presentaron concentraciones fitodisponibles significativamente menores en comparación con el suelo de la balsa sin tratar. Dicha reducción de concentraciones fitodisponibles no predomina en un tratamiento en comparación con el otro (Tabla 5, anexo III). En esta profundidad si comparamos el porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn al final del tiempo experimental (Tabla 8), como ocurrió en la profundidad 0-15 cm, el tratamiento que mejor resultado presentó fue SCBP. Esto puede ser debido a que el compost, al ser una enmienda que pierde eficiencia con más rapidez que otras a lo largo del tiempo, como demostraron Walker et al. (2004), provocaría que algunos de sus compuestos migren acumulándose en profundidades inferiores ayudando a reducir las concentraciones fitodisponibles de los metales.

El tratamiento SCBP, como se detalló anteriormente, presentó en esta profundidad los valores más altos de CT, CEC y pH en comparación con S y STBP. Dichos parámetros influyen directamente en las concentraciones fitodisponibles de metales según autores como Cui et al. (2016), Gusiatin et al. (2016) ó Karer et al. (2015).

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Tabla 8. Profundidad 3, tiempo 3. Porcentaje de la concentración fitodisponible frente a la concentración pseudototal de Cu, Pb, Ni y Zn

% S STBP SCBP

Cu 11,4 18,8 11,5

Pb 1,35 1,51 0,97

Ni 28.8 15,5 19,7

Zn 9,51 10,31 5,85

Los resultados de las correlaciones de Pearson entre CT, CEC, pH y las concentraciones fitodisponibles de los metales estudiados corroboraron lo propuesto por los autores anteriormente citados, con la excepción del Zn (Tabla 9). El compost presentó elevadas concentraciones de Zn por ello probablemente en este caso en particular no se cumpla lo expuesto anteriormente.

Tabla 9. Correlaciones de Pearson entre el pH, capacidad de intercambio catiónica (CEC), carbono total (CT) frente a las concentraciones fitodisponibles de Cu, Pb, Ni, Zn

Cu Pb Ni Zn

CEC -0,948** -0,950** -0,907** 0,852**

pH -0,956** -0,892** -0,933** 0,922**

CT -0,986** -0,969** -0,967** 0,928**

**P˂0,01

7.3.9 Evolución de las concentraciones de Cu, Pb, Ni y Zn en el agua de poro a lo largo de las tres profundidades

El agua de poro se extrajo para conocer las concentraciones de los metales que migrarían a través del suelo, tanto en el suelo de la balsa de decantación sin tratar como una vez aplicados los tratamientos. Para ello, en los cilindros se colocaron muestreadores rhizon en tres alturas diferentes (15 cm, 30 cm, 45 cm). Las tomas se realizaron una vez al mes durante 9 meses. La primera toma se realizó un mes después de la colocación de los cilindros para que los tratamientos estuviesen asentados.

Para facilitar la comprensión de los resultados obtenidos hemos dividido esta apartado en sub-apartados, uno por cada metal.

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7.3.9.1 Evolución de la concentración de Cu en el agua de poro a lo largo de las tres profundidades.

En la profundidad 15cm, tanto la aplicación de compost combinado biochar como de tecnosol mezclado con biochar redujeron de forma significativa las concentaciones de Cu en el agua de poro en comparación con el suelo de la balsa de decantación (Figura 4A).

Figura 4. Concentración de Cu (mg.L−1) en el agua de poro en los controles (S and SS) y tratamientos (STBP and SCBP) en las tres profundidades y a los largo de nueve meses. (n=3, Student’s t test: P< 0.05). Las barras de error representan la desviación estándar. ul: valores por debajo del límite de detección.

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Las concentraciones de Cu en el suelo de la balsa de decantación tratado comienzan a ser detectables a partir de la cuarta toma, lo que nos indica la eficacia de estos tratamientos (Figura 4A). Este descenso de las concentraciones de Cu en el agua de poro se debe a las mejoras, detalladas anteriormente, provocadas en el suelo por el compost combinado con biochar y el tecnosol combinado con el biochar. Una de estas mejoras fue el aumento del pH, el cual esta relacionado con la disminución de las concentraciones de metales en el agua de poro (Oustriere et al., 2017).

El efecto de los tratamientos en las profundidades 30 cm y 45 cm fue muy distinto. En la profundidad 30cm el tratamiento elaborado con compost y biochar redujo significativamente la concentración de Cu desde el tiempo 1 (Figua 4B). Sin embargo, el tratamiento elaborado con tecnosol y biochar no consiguió reducir significativamente estas concentraciones en comparación con el suelo de la balsa hasta pasados siete meses (Figua 4B). Aunque hasta el tiempo 5 las concentaciones de Cu en el agua de poro en el tratamiento STBP fueron mayores que en S, el porcentaje de Cu en el agua de poro frente a las concentaciones pseudototales de Cu nunca superaron el 2%, siendo estos porcentajes en la mayoría de los casos menores al 1% (Tabla 10). Comparando los tratamientos a lo largo del tiempo se observó como, entre el primer y octavo tiempo, el tratamiento SCBP redujo más las concentraciones de Cu, pero en el último tiempo los dos tratamientos igualaron su efecto (Figua 4B).

El mejor comportamiendo del tratamiento elaborado con compost y biochar a la hora de reducir las concentraciones de Cu en el agua de poro pudo deberse al mayor aumento de CEC provocado por este tratamiento (discutido en el apartado 7.3.3) en comparación con el tratamiento elaborado con tecnosol y biochar. La relación de la CEC con la fitodisponibilidad de melates ya ha sido demostrada por Chandra et al. (2014). Por otro lado, como ya se discutió en el apartado 4.3.2, el tratamiento SCBP presentó un mayor contenido de CT en comparación con STBP. Zeng et al. (2015) ya demostraron que el aumento de CT por parte del compost podría estabilizar los metales ya que se ligarían fuertemente al carbono orgánico.

En la profundidad 45 cm, como ocurrió en la profundidad 30cm, el tratamiento elaborado con compost y biochar presentó concentraciones de Cu en el agua de poro significativamente inferiores a las del suelo de la balsa de decantación sin tratar (Figua 4C). Sin embargo, el efecto positivo del tratamiento elaborado con tecnosol y biochar no se detectó hasta el tiempo 8 (Figura 4C). Si observamos el porcentaje de concentración de Cu en el agua de poro frente a las concentraciones pseudototales de Cu, el valor más alto es de 1,20% en el tratamiento STBP a los dos meses de experimento (Tabla 10). A diferencia de lo ocurrido en la produndidad 30cm, en este caso el tratamiento STBP en los últimos dos tiempos presentó concentraciones de Cu en el agua de poro inferiores al tratamiento SCBP.

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Tabla 10. Porcentaje de la concentración de Cu en el agua de poro frente a la concentración pseudototal de Cu

Profundidad Tratamiento Tiempo 1 Tiempo 2 Tiempo 3 Tiempo 4 S - - - - SS - - - - 15 cm STBP - - - - SCBP - - - - S - - - - SS - - - - 30 cm STBP 1,61 - - - SCBP - - - - S - - - - SS - - - - 45 cm STBP 1,20 - - - SCBP - - - - Solo se representan los valores con un porcentaje superior al 1%. Los valores inferiores al 1% se indican con -. Tiempo 1= 2 meses, tiempo 2= 4 meses, tiempo 3= 7 meses, tiempo 4= 8 meses

7.3.9.2 Evolución de la concentración de Pb en el agua de poro a lo largo de las tres profundidades

Las concentraciones de Pb en el agua de poro en la profundidad 15 cm no fueron detectables a lo largo del tiempo experimental una vez se aplicaron los tratamientos sobre el suelo de la balsa de decantación (Figura 5A). En el suelo de la balsa de decantación sin tratar, el Pb sí fue detectable en el cuarto, quinto, sexto, séptimo y noveno tiempo. En esta primera profundidad, que las concentraciones de Pb en el agua de poro no hayan sido detectables una vez se aplicaron los tratamientos, se debe posiblemente a que en la profundidad 0-15cm fue en la que se produjo un mayor aumento del contenido en materia orgánica (Figura 3, anexo IV) y de pH (Tabla 3, anexo IV). Estudios como el realizado por Weng et al. (2001) demostraron que el pH juega un papel crucial en la sorción y solubilidad de metales. Por otro lado, Covelo et al. (2007) propusieron que los suelos con mayor contenido de materia orgánica tienen una mayor capacidad de retención de metales.

En la profundidad 30 cm, una vez se aplicaron los tratamientos SCBP y STBP, las concentraciones de Pb en el agua de poro fueron significativamente inferiores a las del suelo de la balsa de decantación sin tratar (Figura 5B). En esta segunda profundidad, al contrario de lo ocurrido en la primera, las concentraciones de Pb en el agua de poro fueron detectables. Este aumento de las concentraciones del Pb en el agua de poro pudo ser debido a que el aumento de pH (Tabla 3, anexo IV) y CT (Figura 3, anexo IV) fue menor que en la profundidad 15cm. Dichos factores están reclacionados con la sorción y solubilidad de metales (Covelo et al., 2007, Weng

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Discusión general et al. 2001). Las concentraciones de Pb en el agua de poro nunca representaron más del 1% frente a las concentraciones de Pb pseudototal una vez el suelo de la balsa de decantación fue tratado.

Figura 5. Concentración de Pb (mg.L−1) en el agua de poro en los controles (S and SS) y tratamientos (STBP and SCBP) en las tres profundidades y a los largo de nueve meses. (n=3, Student’s t test: P< 0.05). Las barras de error representan la desviación estándar. ul: valores por debajo del límite de detección.

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En los primeros cinco tiempos, el tratamiento más efectivo en este aspecto fue el elaborado con compost y biochar. Por el contrario, en el sexto y séptimo tiempo, el tratamiento elaborado con tecnosol y biochar fue el más efectivo. Por último, en el octavo y noveno tiempo las concentaciones de Pb en el agua de poro no fueron detectables en ambos tratamientos, quedando patente la mejora de la eficacia de ambos tratamientos con el transcurso del tiempo.

El mejor efecto a la hora de reducir las concentraciones de Pb en el agua de poro en la profundidad 45cm se observó en el primer y dos últimos tiempos (Figura 5C). Posiblemente este mejor comportamiento en los dos últimos tiempos se debe a que en esta profundidad el mayor aumento de pH (Tabla 3, anexo IV) y CT (Figura 3, anexo IV) ocurrió al final del experimento. En esta profundidad, 45cm, existe una correlación negativa de Pearson entre el CT y la concentración de Pb en el agua de poro con una r =-0,62 y P˂0,01. Esta correlación negativa corrobora la relación entre la materia orgánica y disminución de las concentaciones de Pb en el agua de poro. Aunque el efecto de los tratamientos no fue notorio hasta los dos últimos tiempos, como ya se indicó, hemos de tener en cuenta que las concentraciones de Pb en el agua de poro supusieron un porcentaje inferior al 1% frente a las concentraciones pseudototales en los meses donde los tratamientos tuvieronmenor efecto.

7.3.9.3 Evolución de la concentración de Ni en el agua de poro a lo largo de las tres profundidades.

La aplicación de los tratamientos SCBP y STBP tuvo un efecto significativo en la disminución de las concentraciones de Ni en el agua de poro en la profundidad 15 cm, con valores siempre por debajo de los del suelo de la balsa de decantación sin tratar (Figura 6A). El buen efecto de estos tratamientos quedó patente si observamos lo ocurrido a lo largo del tiempo. En el tratamiento elaborado con compost y biochar, las concentraciones de Ni no fueron detectables hasta el sexto tiempo y, en el caso del tratamiento elaborado con tecnosol y biochar, ésto ocurrió en el séptimo tiempo (Figura 6A). Esta reducción de las concentraciones de Ni en el agua de poro es importate ya que tanto el tecnosol como el compost presentaban concentraciones pseudototales de Ni superiores al suelo de la balsa de decantación (Tabla 2, anexo III). Si comparamos entre tratamientos no existieron diferencias significativas.

En la profundidad 30 cm entre el segundo y sexto tiempo, el comportamiento de los tratamientos aplicados no presentó un patrón a la hora de reducir las concentraciones de Ni en el agua de poro (Figura 6B). Aun así, los porcentajes de la concentración de Ni en el agua de poro frente a las concentraciones pseudototales de Ni siempre estuvieron por debajo del 1%, salvo la excepción del tratamiento SCBP en el tercer tiempo que fue 1,04% (Tabla 11). En los dos últimos tiempos ambos tratamientos fueron eficaces presentando concentraciones de Ni en el agua de poro significativamente inferiores al suelo de la balsa de decantación sin tratar (Tabla 11). Como

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ocurrió en el caso del Pb comentado en el apartado anterior esta mayor efectividad de los tratamientos en el octavo y noveno tiempo se deba a que en esta profundidad el mayor aumento de pH (Tabla 3, anexo IV) y CT (Figura 3, anexo IV) ocurrió al final de tiempo experimental.

Figura 6. Concentración de Ni (mg.L−1) en el agua de poro en los controles (S and SS) y tratamientos (STBP and SCBP) en las tres profundidades y a los largo de nueve meses. (n=3, Student’s t test: P< 0.05). Las barras de error representan la desviación estándar. ul: valores por debajo del límite de detección.

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En la última profundidad estudiada, 45 cm, la eficacia de los tratamientos SCBP y STBP no se hizo patente hasta el octavo y noveno tiempo, presentando ambos tratamientos, en general, concentraciones de Ni en el agua de poro significativamente superiores al suelo de la balsa de decantación hasta el séptimo tiempo (Figura 6C). Esto se debe posiblemente a que esta profundidad, al estar formada por suelo de la balsa de decantación, el aumento de los parámetros CT y pH por parte de los tratamientos fue progresivo presentado los valores más altos en al final del tiempo experimental, como se observa en la figura 3 y tabla 3 del anexo IV. En este caso, el valor más alto comparando los porcentajes de las concentraciones de Ni en el agua de poro frente a las concentraciones pseudototales de Ni, lo presentó el tratamiento elaborado con tecnosol y biochar a los dos meses de experimento con un 2,01%, porcentaje que disminuyó apartir de ese momento.

Tabla 11. Porcentaje de la concentración de Ni en el agua de poro frente a la concentración pseudototal de Ni. Profundidad Tratamiento Tiempo 1 Tiempo 2 Tiempo 3 Tiempo 4 S - - - - SS - - - - 15 cm STBP - - - - SCBP - - - - S 1,32 - - - SS - - - - 30 cm STBP 1,10 - - - SCBP - - 1,04 - S 2,57 - - - SS - - - - 45 cm STBP 2,01 - - - SCBP - 1,68 - -

7.3.9.4 Evolución de la concentración de Zn en el agua de poro a lo largo de las tres profundidades

Como ocurría con las concentraciones de Cu, Pb y Ni en el agua de poro, en la primera profundidad, en el caso del Zn, el mejor comportamiento se observó en estos primeros 15 cm (Figura 7A). Ambos tratamientos presentaron concentraciones significativamente inferiores al suelo de la balsa de decantación durante todo el experimento. Debemos recordar que el compost presentó 403±3,33 mg/kg de Zn, el tecnosol presentó 340±5,50 mg/kg frente a los 65,4±2,51 mg/kg presentados por el suelo de la balsa de decantación. Esto nos indica la capacidad de fijación de Zn por parte de estos tratamientos. En esta profundidad las concentraciones de Zn del agua de poro están correlacionadas negativamente con el pH y CT ambas con una r=-0,70 y P˂0,01. Lo cual deja patente la importancia del aumento de estos dos factores. Este efecto positivo de los

306

Discusión general tratamientos también se debe al biochar. El biochar tiene muchas propiedades de inmovilización favorables como su estructura microporosa, grupos funcionales activos, un alto pH y capacidad de intercambio catiónico. Además, el biochar tiene una energía adsortiva fuerte para los metales (Inyang et al., 2015).

Figura 7. Concentración de Zn (mg.L−1) en el agua de poro en los controles (S and SS) y tratamientos (STBP and SCBP) en las tres profundidades y a los largo de nueve meses. (n=3, Student’s t test: P< 0.05). Las barras de error representan la desviación estándar. ul: valores por debajo del límite de detección.

307

Discusión general

En las profundidades 30 cm y 45 cm, en general, los tratamientos tuvieron un mejor efecto a final del octavo y noveno tiempo (Figura 7B y C). Aun así, el porcentaje de Zn en el agua de poro frente a la concentración pseudototal de Zn fue inferior al 1%. Hasta el octavo tiempo, el tratamiento elaborado tecnosol y biochar fue menos efectivo en comparación con el elaborado con compost y biochar en la reducción de las concentraciones de Zn en el agua de poro. Como se detalló anteriormente, el tratamiento SCBP presentó concentraciones superiores de CT a lo largo de todo el experimento en las profundidades 15-30 cm y 30-45 cm (Figura 3, anexo IV). Este mayor contenido de CT en el tratamiento SCBP puede explicar su mayor eficacia ya que la correlación de Pearson entre las concentraciones de Zn en el agua de poro y el CT presentó una r=-0,71 y P˂0,01. Además, los porcentajes de las concentraciones de Zn en el agua de poro frente a las concentraciones pseudototales de Zn una vez son aplicados los tratamientos no superaron el 1%. Estos datos indican qué aunque las concentraciones pseudototales de Zn sean altas en el material de partida de los tratamientos la migración de Zn a lo largo del perfil es muy baja.

7.3.10 Evolución de el Factor de translocación (TF) y el Coeficiente de tranferencia (TrC) en las Brassica juncea L. cultivadas sobre STBP y SCBP

En el tercer tiempo es donde, una vez analizados los datos de TF, se observó como en el tratamiento STBP los valores de TF para Pb y Zn superan el valor de 1 (Figura 3, anexo III), valor a partir del cual se considera que una planta es capaz de translocar metales de la raíz a la parte aérea según Baker y Brooks (1989). En cambio, en SCBP, los valores de TF para Pb y Zn nunca superaron el valor 1. Esta diferencia puede ser debida, entre otros factores, a que las concentraciones fitodisponibles de Pb y Zn en el tratamiento STBP eran mayores que en SCBP, sobre todo las de Zn (Tabla 3, anexo III). En ambos tratamientos, al final del tiempo experimental, el valor de TF para Cu y Ni estuvieron por debajo de 1. Según autores como Nouri et al., (2009) y Pinto et al., (2015) si los valores de TF están por debajo de 1 significa que las plantas retienen y fijan los metales en la zona de la raíz jugando un papel importante en la fitoestabilización. Por lo tanto, al final del experimento, en el caso de las Brassica juncea L. cultivadas en STBP, éstas tendrían por un lado una función hiperacumuladora para Pb y Zn, y, por el otro, una función fitoestabilizadora para Cu y Ni. Las Brassica juncea L. cultivadas en SCBP tendrían un papel fitoestabilizador para los cuatro metales estudiados.

Los valores obtenidos de TrC no fueron superiores a 1 en las Brassica juncea L. cosechadas en los tratamientos STBP y SCBP (Figura 4, anexo III), esto es importante ya que según Busuioc et al. (2011), se considera que una planta tiene capacidad fitoextractora según su coeficiente de tranferencia cuando éste presenta valores superiores a 1. Como ocurrió en el caso de los valores de TF, esto no quiere decir que las Brassica juncea L. en nuestro experimento no

308

Discusión general sean funcionales a la hora de recuperar un suelo de este tipo, sino que en vez de tener un papel fitoextractor, lo tendrán fitoestabilizador. Esto ya ha sido propuesto por Kidd et al. (2009) según el cual, si los valores de TrC de una planta están por debajo de 1, significa que dicha planta retiene los metales en la zona de la raíz, presentando una función fitoestabilizadora. Por lo tanto, todos los datos obtenidos de TF y TrC a lo largo del experimento indican que, en general, las Brassica juncea L. cultivadas sobre estos tratamientos tienen un papel fitoestabilizador. Esto es importante ya que la fitoestabilización es considerada desde hace años una de las mejores opciones a la hora de recuperar amplias zonas degradadas como lo son los suelos de mina (Wong 2003).

309

8. Conclusiones

Conclusiones

8. Conclusiones

1- La adición de biochar junto con otras enmiendas orgánicas (compost o tecnosol)

contribuye a la corrección del pH mejor que las enmiendas solas.

2- Al aumentar la profundidad, el tratamiento elaborado con compost, biochar y

Brassica Juncea L. aumentó más el pH que el tratamiento elaborado con tecnosol,

biochar y Brassica Juncea L.

3- Al aumentar la profundidad, la aplicación directa de tecnosol aumentó más el

contenido de carbono que su combinación con biochar.

4- La aplicación combinada de compost y biochar con Brassica juncea L. aumentó

más el contenido de carbono y nitrógeno que la aplicación de tecnosol, biochar y

Brassica juncea L. en prácticamente todas las profundidades y tiempos.

5- La aplicación directa de compost o tecnosol junto con Brassica Juncea L.,

provocaron un mayor aumento de nitrógeno que cuando se combinaban con

biochar.

6- Todos los tratamientos aplicados mejoraron el contenido de nutrientes. La

aplicación de compost, biochar y Brassica Juncea L. aumentó más la disponibilidad

de nutrientes que la aplicación de tecnosol, biochar y Brassica Juncea L.

7- La biomasa de las Brassica Juncea L. fue mayor en los tratamientos que incluían

biochar frente a la aplicación directa de enmiendas. La biomasa fue mayor en las

Brassica Juncea L. cosechadas sobre la combinación de compost y biochar.

8- Los tratamientos que incluían biochar y Brassica Juncea L. redujeron más las

concentraciones fitodisponibles de metales que la aplicación directa de enmiendas

en prácticamente todas las profundidades y tiempos.

9- Al aumentar la profundidad no hubo diferencias en la reducción de las

concentraciones fitodisponibles de metales entre el tratamiento elaborado con

compost, biochar y Brassica Juncea L. y el elaborado con tecnosol, biochar y

Brassica Juncea L.

311

Conclusiones

10- Las Brassica juncea L. cultivadas sobre los tratamientos aplicados presentaron una

buena capacidad fitoestabilizadora, según los valores del Factor de Translocación y

el Coeficiente de Transferencia.

11- La combinación de compost o tecnosol con biochar y Brassica juncea L

disminuyeron las concentraciones de Cu, Pb, Ni y Zn en el agua de poro con

respecto al suelo de la balsa de decantación al final del experimento.

12- En general, la aplicación de combinada de compost con biochar y Brassica juncea L

fue más efectiva en el aumento de pH, carbono, nitrógeno, nutrientes y biomasa

cosechada en el suelo de la balsa de decantación que la aplicación de tecnosol con

biochar y Brassica juncea L.

13- Ha sido demostrada la idoneidad del biochar como complemento en los procesos de

fitorremediación de suelos contaminados por actividades mineras.

312

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