Stabilized municipal sewage sludge addition to improve properties of an acid mine soil for plant growth

1 POTENTIALLY HARMFUL ELEMENTS IN SOIL-PLANT INTERACTIONS 1 2 Stabilized municipal sewage sludge addition to improve properties of an acid mine 3 soil for plant growth 4 5 M.D. Mingorance • S. Rossini Oliva • Benito Valdés • F.J. Pina Gata • Eduardo O. 6 Leidi • I. Guzmán • Aránzazu Peña 7 8 M.D. Mingorance • A. Peña () 9 Instituto Andaluz de Ciencias de la Tierra (CSIC-UGR), Avda. Palmeras, 4, 18100-10 Armilla, Granada, Spain 11 e-mail: [email protected] 12 13 S. Rossini Oliva • B. Valdés • F.J. Pina Gata • I. Guzmán 14 Department of Plant Biology and Ecology, University of Seville, Avda. Reina Mercedes 15 s/n, 41012 Seville, Spain 16 17 E.O. Leidi 18 Department of Plant Biotechnology, IRNAS-CSIC, Avda. Reina Mercedes 10, 41012 19 Seville, Spain 20 21 Unidad Asociada CSIC-US "Estudio Integral del Medio Físico y Biótico" 22 23 24 () Corresponding author:25 Aránzazu Peña 26 Telephone number: 0034 958 230000 27 Fax number: 0034 958 552620 28 e-mail address: [email protected] 29 30 2 Abstract 31 Purpose: Degraded soils, such as those encountered in areas of mine activities, need to be 32 ameliorated by liming to correct soil acidity and by addition of organic inputs to improve 33 soil properties and fertility. 34 Materials and methods: Non amended mine soil and soil amended with stabilized sewage 35 sludge were incubated for 45 d. Soil physicochemical and biological indicators were 36 periodically measured along incubation and other enzyme activities at the end of 37 incubation. In improved soils a study of plant development in 250-g pots was carried out 38 with three vegetal species: tomato, rye grass and ahipa. Germination and mortality rates, 39 biomass production and photosynthetic pigments were measured. 40 Results and discussion: Soil incubation with sewage sludge slightly increased soil pH and 41 led to an enhancement of soil electrical conductivity, organic carbon and dehydrogenase 42 activity, especially for the higher doses (5 and 10%). However soil respiration was more 43 promoted with the 2% dose, pointing to a possible toxic effect of the sludge. At the end of 44 incubation physicochemical and biological properties were in general enhanced. Biomass 45 production was improved in tomato and rye grass by sewage sludge addition (more at the 46 2% dose) whilst ahipa growth was not affected by sewage sludge treatments. Tomato 47 mortality reached 73% with high sludge doses (10%). 48 Conclusions: According to this set of parameters, amendment with SSL of a limed acid 49 mine soil would be considered as a good strategy for soil amelioration in view of plant 50 establishment and development. 51 52 Keywords Ahipa • Mine soil • Organic amendment • Rye grass • Tomato 53 3 1 Introduction 54 The Iberian Pyrite Belt occupies the southwestern corner of the Iberian Peninsula, 55 extending from Seville, in Spain, to the Atlantic Ocean, south of Lisbon, in Portugal, 56 making up a belt of about 230 km in length and 40 km in width (Sáez et al. 1996). It 57 constitutes the world largest massive sulphide deposit (mainly Cu-Pb-Zn), which has been 58 mined since the Metal Age, according to archeometallurgical evidences, but reaching a 59 peak between the 19th and the 20th centuries, when most mines were closed due to 60 exhaustion of the ore (Salkield 1987). 61 The soils in this belt are characterised by high level of acidity, poor physical structure 62 and also contain toxic concentrations of metals and low levels of major plant nutrients 63 (Fernández-Caliani et al. 2009). Even if some communities of pioneer plants colonized this 64 area, it needs to be improved by different measures, which include correction of soil 65 acidity and improvement of soil properties, because the extreme environmental conditions 66 are not suitable to promote plant establishment. Besides, it is essential to assess the 67 efficacy of the implemented measures by evaluating physical, chemical and biological 68 properties, in order to gain a full understanding of constraints and opportunities. 69 Remediation of mine tailings by revegetation is an interesting approach with obvious 70 economic and environmental advantages. However some of the limitations to revegetation 71 on an acid soil include pH values out of physiological values, low nutrient status, low 72 organic matter and microbial activity and low water holding capacity. Addition of organic 73 amendments can increase organic matter, nutrient status, microbial activity and water 74 holding capacity (Jones et al. 2012; González-Ubierna et al. 2012). The use of organic 75 amendments together with liming or other materials rich in carbonates is a way of restoring 76 the ecological function of metal-contaminated sites, given that these approaches improve 77 the physicochemical and biological soil conditions and favour plant growth (Haynes and 78 Swift 1988; Alvarenga et al. 2008, 2009; de Varennes et al. 2010) 79 4 Organic amendments from different sources have been assayed for the improvement of 80 mine soil conditions, from agricultural, industrial or miscellaneous origins (Alvarenga et 81 al. 2008; de Varennes et al. 2010, 2011; Arocena et al. 2012). Furthermore, the use of 82 organic wastes from water treatment plants is an alternative to the disposal of these 83 residues, generated in huge quantities. The applications of these biowastes, whose 84 production is estimated up to 138 million tonnes per year in the EU and with high potential 85 added value, are regulated by a European Directive (Council Directive, ECC 1986) which 86 considers both environmental and soil protection, encouraging the agricultural use of 87 sewage sludge in such a way as to avoid harm to vegetation, animals or humans. In 2007, 88 1.17 Mt of dry urban sewage sludges were produced in Spain, as a result of the treatment 89 of almost 85% of the total residual water, in compliance with European guidelines 90 (Council Directive, ECC 1991). The use of treated sewage sludges provides economic, 91 agronomic and environmental benefits since they are low-cost amendments, have great 92 fertilising ability and are a source of macro (N and P) and micro (Fe, Zn, Cu, etc) nutrients. 93 Additionally they increase soil organic matter content thus improving soil water-holding 94 capacity, microbial activity, and other physical, chemical, and biological properties (Hueso 95 et al. 2012). 96 The aims of this work were (a) to assess the effect of the addition of a stabilized 97 sewage sludge of urban origin on physicochemical and biological properties of a severely 98 degraded mine soil and (b) to study the effect of the amendment on the establishment of 99 three plant species (tomato and rye grass, of agricultural interest and ahipa, a species which 100 could be employed for biofuel production) and therefore their suitability to be used for 101 revegetation. 102 103 2 Materials and methods 104 2.1 Site description and properties of the soil and the amendment 105 5 The soil (NC) was collected in a mine waste situated in the proximity of the village of 106 Nerva, province of Huelva (37 42’ 4.5” N 6 33’ 35.1” W), located in the Iberian Pyrite 107 Belt, which includes one of the largest deposits of pyrite (FeS2) and other metallic and 108 polymetallic sulphides as chalcopyrite (CuFeS2), sphalerite ((Zn,Fe)S) and galena (PbS) 109 (Chopin and Alloway 2007). Soil samples, characterised as sandy loam, were collected at 110 random from the selected site. They were air dried for 2 weeks, sieved through 3-mm for 111 incubation assays, or passed through a 2-mm mesh sieve or ground to 50 m, for analysis 112 following standard methodology. 113 According to X-ray fluorescence analysis SiO2 (46.9%) and Fe (23.0%) and Al 114 (12.7%) oxides represent more than 80% of the soil mineralogical composition. It is a very 115 acid soil (pH 2.4) with low organic carbon content (OC, 1.4%), and high electrical 116 conductivity (EC, 1.3 dS m-1, 1:2.5 ratio); 24% water content at field capacity; HIX 1.16; 117 SUVA (L g-1cm-1) 10.8. Otherwise, this soil contents high total concentrations of potential 118 hazardous elements (mg kg-1) (As, 3951; Cu, 694; Pb, 3976) and also of S (8320). 119 Stabilized sewage sludge (SSL) from the wastewater treatment plant of Granada (SE 120 Spain), was used for the amendment of NC soil. The main properties are: pH 6.9; EC (dS 121 m-1, 1:10 ratio) 2.8; OC (%) 35.5; HIX 0.43. 122 123 2.2 Soil incubation 124 Mine soil (NC) was first limed with Carbocal (Azucarera Ebro), a residue rich in calcium 125 carbonate (83.4%) with an OC content of 5.1%. The limed Nerva soil (NCL) received 126 Carbocal at an equivalent rate of 1.5% (w/w) in CaCO3. After liming the organic waste 127 was applied at 2, 5 and 10% (w/w) (SSL2, SSL5 and SSL10, respectively), corresponding 128 to approximately 40, 100 and 200 Mg ha-1. The mixtures, carried out with air-dried soil and 129 the amendment, were placed in plastic trays covered with aluminium foil to avoid 130 desiccation, their moisture adjusted to 40% of the soil field capacity with deionised water, 131 6 and allowed to stand in the dark at ambient temperature (20 ± 2 ºC), up to 45 d. Water was 132 supplied as required to maintain soil humidity. One subsample was periodically withdrawn 133 (0, 2, 5, 7, 14, 21, 29, 34, 42 and 49 days) for determination of pH, moisture, conductivity 134 and organic carbon (OC) content. Another subsample was kept frozen (-18ºC) until 135 analysis for enzyme activities and soil induced respiration (SIR). 136 137 2.3 Soil induced respiration and microbial biomass C 138 Soil induced respiration (SIR) measurements were performed in an automatic equipment 139 (Trac 4200, Sy-Lab, Gomensoro, Madrid, Spain), after defrozing the samples at ambient 140 temperature. Briefly, ca. 5 g soil was mixed with 50 mg of talc:glucose (10:1 ratio) and 141 weighed into a plastic tube, which was introduced into a measuring cell containing 2 mL of 142 a 2% KOH solution. The tightly closed cell was maintained at 30ºC during 20 h and CO2 143 evolution was monitored every 5 min, through the measurement of solution impedance 144 decrease. Results are expressed as mg CO2 100 g-1 h-1. 145 Soil microbial biomass C (SMBC, mg C 100 g-1 soil) was estimated from the SIR 146 assay. SMBC was calculated from the CO2 generated during 6 h of soil incubation as 147 follows: SMBC = 40.04  CO2 + 0.37 (Anderson and Domsch 1978). 148 149 2.4 Enzyme activities 150 Enzyme activities are used as an index of microbial functional diversity if they reflect 151 changes in microbial activities. Since microbial functional diversity includes many 152 different metabolic processes, a representative set of enzyme activities was assessed: 153 During incubation dehydrogenase activity (DHA), proposed as a measure of overall 154 microbial activity, was determined by incubating soil samples during 20 h at 25°C with 0.2 155 mL of 0.4% 2-p-iodophenyl-3 p-nitrophenyl-5 tetrazolium chloride as a substrate. The 156 7 iodonitrotetrazolium formazan (INTF) formed was measured spectrophotometrically at 490 157 nm, according to García et al. (1997). 158 At the end of the incubation period various enzymes were analyzed: enzymes 159 participating in the C cycle (-glucosidase), N cycle (protease), P cycle (alkaline 160 phosphatase) and S cycle (arylsulphatase). The enzyme activities -glucosidase (Glu), 161 alkaline phosphatase (AlkP) and arylsulphatase (Aryl) were spectrophotometrically 162 quantified at 400 nm by estimating the p-nitrophenol (PNP) released by incubating the soil 163 with a substrate containing a p-nitrophenyl moiety (Tabatabai and Bremner 1970; Ladd 164 and Butler 1972; Tabatabai 1994) and expressed as g PNP g-1 h-1. Protease activity (Pro) 165 determined the released tyrosine reacted with Folin-phenol reagent measuring at 700 nm 166 by spectrophotometry (Ladd and Butler 1972). This activity is expressed as g tyrosine g-1 167 h-1. All enzyme activities were determined in defrozen samples in triplicate and expressed 168 on an oven-dried (105ºC) soil basis. 169 170 2.5 Analytical methods 171 The soil particle size distribution was determined by sieving and sedimentation, applying 172 the Robinson’s pipette method after organic matter had been removed with H2O2, using 173 sodium hexametaphosphate as dispersing agent. Field capacity was obtained from water 174 retention of disturbed soil samples using ceramic pressure plates at an air pressure of 0.03 175 MPa. The pH and EC determinations were carried out in sample/deionised water 176 suspensions 1/2.5 (w/v) for soil and mixtures of soil and sludge and 1/10 (w/v) for sludge. 177 Organic C (OC) content was determined by a modified Walkey and Black method 178 (Mingorance et al. 2007). The humification index (HIX) (Zsolnay 2003) and the speci®c 179 UV absorbance (SUVA, L g1 cm1) (Hernández-Soriano et al. 2011) were determined in 180 sample/deionised water suspensions 1/4 (w/v) at the end of the incubation period. Samples 181 8 were analysed in triplicate. Estimation of metal content was accomplished by X-ray 182 fluorescence analysis. 183 184 2.6 Pot experiments 185 Three plant species were selected for the experiments: two of agricultural interest, tomato 186 (Lycopersicon esculentum Mill.) and rye grass (Lolium perenne L.), and ahipa 187 (Pachyrhizus ahipa (Wedd.) Parodi), whose tuberous root could be used for biofuel 188 production. The experiment was carried out in pots of 250 g and under greenhouse 189 conditions (average temperature 21.8°C and humidity 67%). Non-amended limed soil 190 (NCL) as a control and NCL amended with stabilized SSL at 2 and 10% (SSL2; SSL10) 191 were the treatments tested. 192 In each pot the number of seeds was planted according to previous germination assays. 193 The seeds showed the following germination rate: tomato 85%, rye grass 95% and ahipa 194 70%. A total of ten plants per pot were cultivated. Tap water was periodically added to 195 maintain soil field capacity. In order to reduce soil compactness, 40 g of glass beads (4 196 mm) were mixed in each pot. 197 Germination and mortality rates, biomass production and photosynthetic pigments were 198 measured. Germination was monitored at the beginning and mortality at the end of the 199 assays. The experiment was carried out during 18 days for rye grass and for one month for 200 tomato and ahipa harvesting plants at vegetative stage. Fresh weight of the plant aerial 201 vegetative part was only measured at the end of the experiment. Photosynthetic pigments 202 were determined using a spectrophotometer (Thermo, Helios Gamma). A piece of leaf was 203 taken and pigments were extracted with pure methanol for 24 hours and then the extract 204 was measured. The concentrations of Chlorophyll a (Chl. a), b (Chl. b) and the sum of leaf 205 carotenoids were calculated with the following equations given for pure methanol, where 206 9 the pigment concentrations are given in µg mL-1 extract solution (Lichtenthaler and 207 Buschmann 2001) 208 209 210 211 212 213 2.7 Statistical treatment of the data 214 Exploratory analysis was carried out to check normality of the data sets. Differences 215 between treatments were determined by ANOVA of normal data sets and Kruskal-Wallis 216 test of non-normal data sets. Otherwise, the post-hoc Tukey-t or Fisher's LSD (least 217 significant difference) tests were used for comparison of several means. Comparison 218 between two sample means was performed by t-test or Mann-Whitney test. The 219 relationship between variables was performed by either correlation or regression analysis. 220 Hierarchical cluster analysis was used to arrange the soil and plant properties into groups 221 using the Ward’s method as linking algorithm and the square Euclidean distance as 222 similarity measurement. The clustering results were shown in a dendogram to provide 223 grouping of variables. 224 Values with p < 0.05 were considered significant. SPSS v.17.0 (Illinois, USA) was 225 used for statistical data analysis. 226 227 3 Results and discussion228 3.1 Evolution of soil physicochemical properties along incubation 229 Addition of Carbocal as a liming agent effectively raised soil pH with an average value of 230 6.8 ± 0.18 for the whole assayed period. The increase in pH as a result of liming has been 231 linked with a change in the charge characteristics of soil OC (Curtin and Smillie 1983; 232 [Chlorophyll a] = (16.75 * A665,2) – (9.16 * A652,4) [Chlorophyll b] = (34.09 * A652,4) – (15.28 * A665,2) [Carotenoids] = ((1000 *A470) – (1.63 * [Chlorophyll a]) – (104.96 * [Chlorophyll b])) 221 16 application, tomato stunted growth and later plant death might have resulted by the 388 significant increase in soil EC (Table 1) (the species is more sensitive to salinity at 389 germination and early seedling stage, see Foolad 1996). Inhibition of plant growth in soils 390 of high salinity has been reported for other vegetal species when added with sewage sludge 391 or other organic amendments (Gascó and Lobo 2007; Pardo et al. 2011). However, other 392 effects should not be discarded like overcoming toxicity thresholds either from metals in 393 the sludge or in the contaminated soil. In tomato, the SSL treatment had a positive effect 394 on photosynthetic pigments (see Table 2), being significantly higher in plants growing in 395 amended soil as compared to non amended soil (p < 0.05) without showing significant 396 differences between doses (p > 0.05). Estimation of chlorophyll content is often 397 accomplished to assess the impact of most environmental stresses as the pigment content is 398 linked to toxicity/deficiency symptoms and photosynthetic plant productivity (Gupta and 399 Sihna 2007). Our results are in agreement with earlier reports (Gupta and Sihna 2007; 400 Singh and Sihna 2005) and may be attributed to the improved bioavailability in the soil of 401 elements required for chlorophyll biosynthesis or the additional supply of nutrients from 402 the sludge. Carotenoids, protective pigments associated to chlorophylls, increase 403 sometimes under stress conditions (Kenneth et al. 2000; Rossini et al. 2010) and the results 404 suggest the metal contamination of the soil and the metal contribution of sludge 405 application. 406 An exploratory cluster data analysis (see Fig. 3), carried out to discover similarities 407 among the main soil properties and the plant growing indicators, revealed that plant 408 mortality was related with soil properties and the dose of SSL; the biomass and pigment 409 content were related with soil respiration, an indicator of soil fertility; finally, the third 410 group is related with the quality of DOC but seems not to be related with plant growth. 411 There is clear evidence that addition of SSL had a strong effect on plant establishment and 412 at the same time a dose increase resulted in an improvement of the soil biological activity 413 17 and microbial efficiency and, depending on the species, of the plant growth. 414 Finally, if we focus on individual plant species, ahipa and rye grass grow well up to 415 SSL10, while a dose > 2% negatively affects tomato establishment. 416 417 4 Conclusions418 The addition of organic amendments to degraded soils, such as those from acid mining 419 areas, should be selected taking into consideration not only soil fertility, but also soil 420 ability to protect the underlying saturated zone from pollution and the ability to constitute 421 an optimum ecosystem for the introduction of appropriate plants for revegetation purposes. 422 Addition of SSL from urban treatment plants resulted in an initially enhanced soil OC, 423 though it decreased with time as a result of sludge stabilization, and a general increase of 424 biological and biochemical parameters (enzymatic activities and soil respiration). 425 According to this set of parameters, soil amended with SSL would be considered as a good 426 candidate for soil revegetation and plant establishment and development. All the species 427 tested (tomato, rye grass and ahipa), with different economic and agricultural interest, 428 could be used for revegetation after liming this soil for pH correction and the use of the 429 amendment at 2%, which implies less economic investment, might be recommended for 430 the establishment of tomato plants. However to confirm the observed effects attained with 431 the present approach, based on a screening carried out with the three plant species in small 432 pots, a future test under field conditions will be required. 433 434 Acknowledgements Thanks to Proyecto de Excelencia, Junta de Andalucía (P10-435 RNM5814), cofinanced with European FEDER-FSE funds. The technical assistance of 436 Isabel Cañadas is gratefully acknowledged. EMASAGRA is thanked for the kind supply of 437 stabilized sewage sludges. 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Geoderma 569 113:187-209 570 571 572 24 Figure caption sheet 573 574 Fig. 1 Evolution during incubation of electrical conductivity (left) and soil organic carbon 575 (right) for limed Nerva soil (NCL) amended with stabilized sewage sludge (SSL) at 0, 2, 5 576 and 10% 577 578 Fig. 2 Evolution during incubation of soil induced respiration (left) and dehydrogenase 579 activity (right) for limed Nerva soil (NCL) amended with stabilized sewage sludge (SSL) at 580 0, 2, 5 and 10% 581 582 Fig. 3 Dendrogram for hierarchical clustering of soil and plant variables583 Figure 1. Evolution during incubation of electric conductivity (left) and soil organic carbon (right) for limed Nerva soil (NCL) amended with stabilized sewage sludge (SSL) at 0, 2, 5 and 10%. 0 1 2 3 4 5 010 20 30 40 50 Incubation time (d) EC (dS m-1) 0 1 2 3 4 5 010 20 30 40 50 OC (%) SSL0 SSL10 SSL2 SSL5 SSL0 SSL10 SSL2 SSL5 0 1 2 3 4 5 010 20 30 40 50 Incubation time (d) EC (dS m-1) 0 1 2 3 4 5 010 20 30 40 50 OC (%) SSL0 SSL10 SSL2 SSL5 SSL0 SSL10 SSL2 SSL5