Evaluation of the phytostabilisation efficiency in a trace elements contaminated soil using soil health indicators

Journal of Hazardous Materials 268 (2014) 68–76

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Evaluation of the phytostabilisation efficiency in a trace elements contaminated soil using soil health indicators T. Pardo a,∗ , R. Clemente a , L. Epelde b , C. Garbisu b , M.P. Bernal a a Department of Soil and Water Conservation and Organic Waste Management, CEBAS-CSIC, Campus Universitario de Espinardo, P.O. Box 165, 30100 Murcia, Spain b Department of Ecology and Natural Resources, Soil Microbial Ecology Group, NEIKER-TECNALIA, Berreaga 1, 48160 Derio, Spain

h i g h l i g h t s • • • • •

Microbial and ecotoxicological parameters were evaluated in a phytostabilised soil. Amendments enhanced soil habitat function, nutrients cycles and reduced toxicity. Nutrients stimulated the growth, activity and diversity of soil microorganisms. Soil health increased after addition of amendments and A. halimus establishment. Using compost with A. halimus is suitable for semi-arid mine soils remediation.

a r t i c l e

i n f o

Article history: Received 21 October 2013 Received in revised form 18 December 2013 Accepted 3 January 2014 Available online 8 January 2014 Keywords: Heavy metals Arsenic Organic amendments Ecotoxicological bioassays Soil functional diversity

a b s t r a c t The efficiency of a remediation strategy was evaluated in a mine soil highly contaminated with trace elements (TEs) by microbiological, ecotoxicological and physicochemical parameters of the soil and soil solution (extracted in situ), as a novel and integrative methodology for assessing recovery of soil health. A 2.5-year field phytostabilisation experiment was carried out using olive mill-waste compost, pig slurry and hydrated lime as amendments, and a native halophytic shrub (Atriplex halimus L.). Comparing with non-treated soil, the addition of the amendments increased soil pH and reduced TEs availability, favoured the development of a sustainable vegetation cover (especially the organic materials), stimulated soil microorganisms (increasing microbial biomass, activity and functional diversity, and reducing stress) and reduced direct and indirect soil toxicity (i.e., its potential associated risks). Therefore, under semi-arid conditions, the use of compost and pig slurry with A. halimus is an effective phytostabilisation strategy to improve soil health of nutrient-poor soils with high TEs concentrations, by improving the habitat function of the soil ecosystem, the reactivation of the biogeochemical cycles of essential nutrients, and the reduction of TEs dissemination and their environmental impact. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Soil contamination by trace elements (TEs) due to mining activities generally involves the modification of its ability to sustainably develop its potential ecological functions and may affect adjacent ecosystems, i.e. alter soil functioning and health [1,2]. This is the case of the mining area of the Sierra Minera of La Unión-Cartagena (Murcia, SE Spain), where the intense mining activity carried out for over 2500 years has given rise to highly contaminated soils with unfavourable conditions for plant growth (high levels of TEs, low organic matter and nutrient contents, high salinity, poor physical structure, etc.), most of them unable to support a vegetation cover to protect the soils from wind and water erosion [3,4]. To

∗ Corresponding author. Tel.: +34 968396264; fax: +34 968396213. E-mail addresses: [email protected], [email protected] (T. Pardo). 0304-3894/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2014.01.003

face this problem, phytostabilisation, based on the use of native tolerant plants and agricultural practices, could be a valuable and cost-effective option to remediate this type of soils [4], reducing in situ the toxicity and dissemination of TEs while promoting soil natural processes [5]. The assessment of the recovery of soil health after a remediation procedure will allow the evaluation of its effectiveness and, thus, to optimise its application in future restoration processes [2,6]. The use of indicators related to soil physicochemical, ecological and ecotoxicological characteristics, which combine laboratory tests and field monitoring, seems to be a suitable screening methodology of soil health [7,8], allowing the evaluation, not only of soil functioning, but also of its associated risks. Parameters related to the soil microbial ecology, especially those dealing with the size, activity and diversity of soil microbial communities, are considered as potentially sensitive, early and effective indicators of soil health [9,10], and have been proposed as useful

T. Pardo et al. / Journal of Hazardous Materials 268 (2014) 68–76

tools to evaluate and assess the effectiveness of the remediation of TEs contaminated soils [11–13]. For example, increases in soil biomass-C and N [4], hydrolase activities related with the major nutrient cycles [1,14,15], mineralisation of N [6], microbial respiration [2] or diversity indexes [6,16,17] have been reported in different TEs contaminated soils after a remediation process, reflecting the amelioration of soil functioning. Nevertheless, just a few studies in highly TEs contaminated mine spoil soils that integrate these indicators with ecotoxicological parameters can be found in the literature (e.g. [18–20]). Ecotoxicological assays (direct and indirect tests) can reflect soil toxicity by showing the effects of the interactions between the contaminants, the soil matrix and the living organisms in it [21,22]. In TEs contaminated soils, ecotoxicological tests have generally been applied alone or in combination with chemical analyses to evaluate the soil’ potential ecological risks [22,23], but this approach does not provide a global view of the remediation process effect on soil health. The aim of this work was to evaluate the efficiency of the application of organic wastes and lime as soil amendments, in combination with a native plant species, on the phytostabilisation of a mine TEs contaminated soil from the Sierra Minera of La Unión-Cartagena by studying the recovery of soil health. The short-term effects of these treatments on chemical properties of TEs contaminated soils and their evolution under field conditions have been reported in previous studies [4,13], but their effectiveness to recover soil health is evaluated at the field conditions in the present paper. After 2.5 years of field experimentation, the success of the phytostabilisation process on soil health recovery was evaluated through the study of the physicochemical properties of the soil and soil solution (extracted in situ), soil microbial parameters, and direct and indirect ecotoxicological bioassays. 2. Materials and methods 2.1. Experimental set-up A field phytostabilisation experiment was located in a mine spoil soil contaminated with trace elements (Table 1) near the village of El Llano del Beal (Murcia, SE Spain). Two organic materials and an inorganic liming substance were used as soil amendments: mature olive mill-waste compost (60 t ha−1 ), fresh pig slurry (60 m3 ha−1 ) and hydrated lime (2.3 t ha−1 ). Four replicates per treatment were randomly distributed in 12 plots of 2 m2 (2 m × 1 m) leaving 0.5 m aisles between plots. In each plot, one sub-plot of 1 m2 was planted with the native halophytic shrub Atriplex halimus L., and another

Table 1 Characteristics of the soil and the organic amendments. Characteristics

Soil

Compost

Pig slurrya

pH EC (dS m−1 ) CEC (cmolc kg−1 ) OM (%) TOC (g kg−1 ) Total-N (g kg−1 ) Total-P (g kg−1 ) Available-P (g kg−1 ) Available-As (mg kg−1 ) As (mg kg−1 ) Cd (mg kg−1 ) Cu (mg kg−1 ) Fe (g kg−1 ) Mn (mg kg−1 ) Pb (mg kg−1 ) Zn (mg kg−1 )

6.2 ± 0.1 2.7 ± 0.01 6.9 ± 0.3 0.2 ± 0.01 1.3 ± 0.1 0.4 ± 0.05 na 0.07 ± 0.04 0.39 ± 0.01 664 ± 28 19 ± 1 193 ± 8 108 ± 1.1 4073 ± 368 10,188 ± 97 9686 ± 251

8.8 ± 0.01 6.1 ± 0.2 na 73.1 ± 0.3 438.6 ± 7.9 31.7 ± 0.8 4.9 ± 0.1 <0.01 na 4.9 + 0.1 <0.01 48 ± 0.5 1.62 ± 0.1 92 ± 1 36 ± 2 141 ± 4

7.8 ± 0.01 24.3 ± 0.01 na 1.05 ± 0.07 7.9 ± 0.04 3.9 ± 0.01 0.5 ± 0.1 <0.01 na <0.01 <0.01 12 ± 0.4 0.045 ± 0.004 11 ± 1 0.9 ± 0.2 116 ± 2

a

Expressed in fresh weight (moisture = 97.4%); na: not analysed.

69

one was left without plants (it was assumed that the roots from planted subplots did not affect significantly unplanted ones). Full details of the experimental set-up and of the evolution of soil, plants and pore water properties during the first two years of experimentation have been widely described in Clemente et al. [4]. Two and a half years after the transplanting of A. halimus plants, topsoil (0–20 cm) from each sub-plot and from four points randomly distributed just outside the experimental plots (as control-unamended soil) were sampled. Soils were sieved to <2 mm; subsequently, one sub-sample was air dried and another was kept refrigerated at 4 ◦ C at its field moisture content for the microbial analyses. Additionally, 10 kg of soil from a “model” plot per treatment and from the four control external points were taken for the ecotoxicological evaluation. The model plots were selected in terms of their pH value (all about 7.5), in order to reduce the influence of the heterogeneity observed in the experimental area. Finally, soil pore water was collected in situ from the plots at a depth of 15 to 20 cm, using FLEX type “Rhizon” samplers (Eijkelkamp Agrisearch Equipment, The Netherlands) [4]. 2.2. Analytical methods 2.2.1. Chemical analyses The physicochemical characteristics of the soils and the organic amendments (Table 1) were determined according to the methods described in Pardo et al. [13]. Soluble and exchangeable TEs concentrations (0.1 M CaCl2 extracts (1:10 w/v)) were determined by AAS (Unicam 969 AAS, Thermo Elemental). Soil available-As (0.5 M NaHCO3 extracts (1:10 w/v)) was measured by hydride generation atomic fluorescence spectrometry (PSAnalytical Millennium Excalibur, UK). Water soluble C and N (CW and NW ) were determined in water extracts (1:10 w/v) by an automatic microanalyser (TOCV CSN + TNM-1 Analyzer, Shimadzu, Japan). The concentrations of available-K (10 mM NaNO3 extracts (1:5 w/v)) were determined by ICP-OES (Iris Intrepid II XDL, Thermo Scientific). All the analyses were done at least in duplicate and adjusted to values for ovendried soil (105 ◦ C for 24 h). Analytical errors (standard error) were below 2%. Trace element concentrations in the soil pore water were determined by ICP-OES (Thermo Scientific). Dissolved organic-C (DOC) and N were determined in a TOC-V analyzer (Shimadzu). 2.2.2. Microbial parameters Microbial biomass-C (BC ) and -N (BN ) were measured according to the methods described in Pardo et al. [13]. Basal respiration was determined after incubation of soil during 10 days (in the darkness at 26 ◦ C) with a trap of 0.1 M NaOH for retention of the CO2 evolved, by titration of the residual NaOH with HCl in an excess of BaCl2 [24]. ␤-Glucosidase, urease, acid phosphatase, arylsulphatase, and dehydrogenase activities were determined according to Tabatabai [25], Kandeler and Gerber [26] and Taylor et al. [27], as described in Epelde et al. [6,17]. Fluorescein diacetate (FDA) activity was determined using tris(hydroxymethyl)aminomethane (THAM; 100 mM, pH 7.6) as a buffer and FDA (0.2%, w/v) as substrate at 25 ◦ C for 15 min, stopping the reaction with acetone and measuring spectrophotometrically the absorbance at 490 nm. Communitylevel physiological profiles (CLPPs) were determined using Biolog EcoPlatesTM according to Epelde et al. [6]. Soil samples were extracted with sterilised Milli-Q ultrapure water (1:10 w/v) for 1 h. A 1:100 dilution of the extract was inoculated onto the plates, which were incubated at 26 ◦ C for 7 days, and colour development was read periodically at 595 nm using a microplate reader (Anthos Zenyth 3100). For each reading time, average well colour development (AWCD) was determined by calculating the mean of every well’s absorbance value. Metabolic richness (S, the number of substrates with an absorbance value > 0.15) and Shannon’s

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diversity index (H = −pi log2 pi ; where pi is the ratio of the corrected absorbance value of each well (from 1 to S) to the sum of absorbance values of all wells) were calculated at 66 h incubation time (when the highest rate of microbial growth was observed in the Biolog EcoPlatesTM ). 2.2.3. Ecotoxicity bioassays Direct and indirect test were performed using the soils or their saturation extracts and a range of dilutions (made with artificial OCDE soil [28] or a diluent prepared according to the corresponding method) appropriate to calculate the EC50 values (soil or extract concentration – w/w or v/v% – at which a lethal or toxic effect on 50% of the population of organisms can be observed). Whenever possible, EC50 values were calculated applying a linear regression analysis between the logarithm of the % of sample concentration versus % of the effect (growth inhibition, and mortality or luminescence inhibition). Plant growth tests with Zea mays L. (maize) and Lactuca sativa L. (lettuce) (a monocotyledonous and a dicotyledonous species, respectively) were carried out as a direct acute toxicity bioassay according to a standardised protocol [29] with some modifications. One seed for Z. mays or three seeds for L. sativa were sown in plastic pots with 100 ± 5 g of the corresponding test soil (at 70% WHC). Ten (maize) or five (lettuce) replicates of each treatment were performed for each species, and were maintained in an environment-controlled chamber (25/18 ◦ C, 58/70% moisture, 11/13 h light/darkness). Fourteen or 21 days after 50% of seedlings of Z. mays and L. sativa, respectively, had emerged in the control soil (0%), plants were harvested and weighed. Growth was expressed in terms of plant dry weight (48 h at 60–70 ◦ C). The luminescence inhibition of Vibrio fischeri test was performed according to ISO 11348-2 [30], using the BioToxTM Kit (Aboatox Oy, Finland). The decrease of luminescence was measured in duplicate after 15/30 min contact of a given volume of the sample to be tested with a suspension of the luminescent bacteria at 15 ± 1 ◦ C. The inhibition of luminescence was calculated through the following equation: In h% = 100–100 × (ITt /(KF × IT0 )), where KF = ICt /IC0 (correction factor), IC0 and ICt the luminescence intensity of the control at the start and after 15 min or 30 min, and IT0 and ITt the luminescence intensity of the sample at the start and after 15 or 30 min, respectively. The aquatic crustaceous Thamnocephalus platyrus immobilisation test was developed using the Thamnotoxkit FTM (MicroBioTests Inc., Belgium). Ten individuals, hatched from cysts 24 h prior to the start of the test, were exposed in triplicate at each level of dilution for 24 h, at 25 ± 2 ◦ C and in the darkness. Immobilisation was recorded after 24 h of exposure and compared with the control. Seed germination bioassays were performed as described in Pardo et al. [13] adapted from Zucconi et al. [31], using soil water extracts (1:10 w/v [32]), and Lepidium sativum L. (cress) and L. sativa L. (lettuce) seeds. In order to test the effect of salinity on seed germination, a CaCl2 solution with the same electrical conductivity as the soil extract was also used. Percentages of seed germination (G), root elongation (R) and germination index (GI = [G × R]/100) were calculated after exposure to the extracts for 3/5 days at 21/17 ◦ C for cress and lettuce, respectively. 2.3. Statistical analysis Data were subjected to ANOVA analysis, and differences between means were determined using Tukey’s test. Normality of variances was tested by the Kolmogorov–Smirnov test before determining the ANOVA and transformed to accomplish normality when necessary. Simple correlations between the different variables were calculated with the Pearson’s test. Principal component analyses (PCAs) were run with all the parameters to enlighten

general tendencies. All the statistical analyses were made with SPSS Version 19.0 software (SPSS Inc.). 3. Results and discussion 3.1. Soil physicochemical properties and TEs solubility After 2.5 years of experimentation, the addition of the amendments led to similar values of soil pH, remaining near neutrality in all the treatments (Table 2). However, a certain heterogeneity of soil and pore water pH was observed (Table 2), with some parts of the plots showing slightly acidic pH values. This was a consequence of the mineralogical heterogeneity of the soil and of preferential rain water flow paths formed in the soil [4]. Soil salinity was elevated in all the plots (2.7–3.0 dS m−1 ), but slightly lower in all the amended sub-plots compared to unamended control soils (CT: 3.5 dS m−1 ; p > 0.001). Water soluble organic C (CW ) concentration in soil was higher in soils treated with compost than in control or limed treatments, despite the long time elapsed since its addition to the soils (p < 0.001, Table 2). Additionally, soil CW increased with the presence of A. halimus (TxPl: p < 0.001) as a consequence of the incorporation of rests of leaf litter, the production of organic compounds by plant roots, and the microbial activity stimulation [2,33]. This input compensated the lower persistence of OM from pig slurry, reaching similar values in planted sub-plots that in composttreated soils. However, this “plant effect” was not observed in hydrated lime-amended soils, because the plants did not grow well in those plots. The addition of organic amendments slightly increased water soluble N (NW ) concentrations in the soils with respect to the controls and only compost enlarged the available-P (p < 0.001; p < 0.01, Table 2). These concentrations, together with the available-K, also increased in the presence of the plants (TxPl: p < 0.01; p < 0.05; Pl: p < 0.05, respectively), suggesting plant litter degradation in the soils throughout the experiment. However, greater dissolved N concentrations in pore water were found in limed soils with respect to the organic treatments (p < 0.05). This may be associated to the lower plant and microbial development in the lime treated soils, as the N present in pore water is mostly inorganic (NO3 − N) and easily available for the plants and soil microorganisms. High CaCl2 -extractable Zn concentrations were found in all treatments, but in pore water only for compost and lime (with plants) treatments (Table 2). The concentrations of CaCl2 extractable Cd, Cu, Mn and Pb, and of NaHCO3 -extractable As in soils and dissolved (pore water) were rather low with the exception of the control (Table 2), reflecting low TEs solubility in these soils [4]. However, elevated levels of these TEs were found in soil and pore water samples from certain plots, coinciding with the acidic parts of the plots mentioned above, as indicated by the negative correlations observed between soil pH and TEs extractable concentrations (pH-Cd: r = −0.879, p < 0.001; pH-Mn: r = −0.800, p < 0.001; pH-Zn: r = −0.881, p < 0.001, n = 28). As a consequence, no significant effects for treatment or plant presence were observed, as reported by Clemente et al. [4], in agreement with the results obtained in an incubation experiment carried out with the same soil and the same amendments [13]. 3.2. Soil microbial parameters as indicators of soil health 3.2.1. Soil microbial biomass After 2.5 years of the experiment, soils treated with compost and pig slurry (with plants) showed statistically larger concentrations of soil microbial biomass-C (BC ) and -N (BN ) (p < 0.001 and

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71

Table 2 Physicochemical properties of soil and pore water samples from the different treatments at the end of the experiment (mean ± SE; soil: n = 4, pore water: n = 2). Cu <1 mg kg−1 and <0.01 mg L−1 .

Soils H. lime-no plant H. lime-plant Compost-no plant Compost-plant Pig slurry-no plant Pig slurry-plant Control T Pl TxPl Pore water H. lime-no plant H. lime-plant Compost-no plant Compost-plant Pig slurry-no plant Pig slurry-plant T Pl TxPl

pH

CW

NW

Avail.-P

Avail.-K

Extract.-As

Extract.-Cd

Extract.-Mn

Extract.-Pb

Extract.-Zn

7.0 ± 0.4 7.1 ± 0.5 6.9 ± 0.3 7.0 ± 0.6 7.2 ± 0.2 7.1 ± 0.7 6.2 ± 0.5

g kg−1 17.1 ± 2.3bc 22.1 ± 4.3bc 72.7 ± 13.2a 86.4 ± 20.2a 24.2 ± 2.4bc 53.3 ± 13.9ab 14.1 ± 1.1c

g kg−1 0.05 ± 0.01bc 0.04 ± 0.01c 0.18 ± 0.03a 0.16 ± 0.06ab 0.06 ± 0.01abc 0.16 ± 0.05ab 0.03 ± 0.01c

mg kg−1 0.22 ± 0.07 b 0.23 ± 0.06 b 1.01 ± 0.39 ab 1.73 ± 0.92 a 0.71 ± 0.20 ab 0.87 ± 0.22 ab 0.20 ± 0.07 b

mg kg−1 24 ± 6 40 ± 17 52 ± 15 133 ± 85 24 ± 11 169 ± 75 23 ± 4

mg kg−1 1.10 ± 0.26 0.92 ± 0.13 1.23 ± 0.43 1.28 ± 0.48 1.04 ± 0.05 1.19 ± 0.34 0.60 ± 0.26

mg kg−1 2.6 ± 1.80 2.3 ± 1.46 4.7 ± 3.26 4.1 ± 4.06 3.3 ± 1.29 2.4 ± 1.86 6.2 ± 2.30

mg kg−1 21.6 ± 23.0 15.7 ± 15.4 57.6 ± 50.3 48.8 ± 57.5 5.3 ± 3.1 29.4 ± 31.0 83.7 ± 81.3

mg kg−1 6.3 ± 5.19 3.4 ± 2.08 3.3 ± 1.40 3.4 ± 2.51 3.1 ± 1.06 13.6 ± 13.8 63.3 ± 51.1

mg kg−1 213 ± 234 148 ± 160 321 ± 319 298 ± 372 72 ± 45 183 ± 199 518 ± 370

***

***

**

ns

ns

ns

*

***

**

*

ns

ns ns ns

ns ns ns

ns ns ns

*

*

ns ns

ns ns ns

6.7 ± 0.4 5.8 ± 0.5 6.5 ± 0.3 6.3 ± 0.5 6.5 ± 0.2 7.2 ± 0.0

mg L−1 10.4 ± 0.5 13.4 ± 2.2 17.2 ± 3.2 15.0 ± 2.3 16.5 ± 0.0 10.2 ± 0.1

mg L−1 8.1 ± 3.9 4.0 ± 0.7 1.4 ± 0.8 1.7 ± 0.9 1.2 ± 0.1 1.2 ± 0.1

<0.01 <0.01 <0.01 <0.01 <0.01 <0.01

– – – – – –

mg L−1 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

mg L−1 0.06 ± 0.07 0.45 ± 0.27 0.29 ± 0.19 0.22 ± 0.19 <0.01 <0.01

mg L−1 1.0 ± 0.9 15.4 ± 15.3 3.5 ± 3.2 4.1 ± 3.1 <0.01 <0.01

mg L−1 0.10 ± 0.11 0.43 ± 0.29 0.23 ± 0.16 0.22 ± 0.13 0.03 ± 0.02 0.10 ± 0.10

mg L−1 19 ± 17 138 ± 97 75 ± 53 71 ± 47 1.1 ± 0.4 0.5 ± 0.4

ns ns ns

ns ns ns

**

– – –

– – –

– – –

ns ns ns

ns ns ns

ns ns ns

*

ns ns ns

ns *

ns ns

Mean values denoted by the same letter in a column do not differ significantly according to Tukey’s test (p > 0.05). –, not determined. In the ANOVA, T, treatment; Pl, plant (A. halimus). * p < 0.05. ** p < 0.01. *** p < 0.001.

p < 0.01; Table 3) than control (up to 10-fold) and limed soils. The presence of plants acted in a different way for each treatment (TxPl: p < 0.001 and p < 0.001), having a certain effect in pig slurrytreated soils, where BC and BN concentrations were significantly higher in planted sub-plots with respect to their corresponding unplanted ones. The long term supply of essential nutrients by the compost may have been the key factor for the observed higher microbial development, as suggested by the positive significant correlations found between both parameters (BC and BN ) and CW , NW , available-P and -K concentrations (all p < 0.001; Table A.1, Supporting Information). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2014.01.003.

Table 3 Soil microbial biomass-C (BC ) and -N (BN ) and diversity indexes (S, H ) at the end of the experiment (mean ± SE; n = 4). Treatments

BC (mg kg−1 )

BN (mg kg−1 )

S

H

H. lime-no plant H. lime-plant Compost-no plant Compost-plant Pig slurry-no plant Pig slurry-plant CT

24 ± 5.2c 20 ± 6.6c 167 ± 58a 250 ± 94a 29 ± 4.9bc 91 ± 31ab 16 ± 3.1c

0.3 ± 0.1c 0.2 ± 0.1c 10 ± 6a 20 ± 15a 0.4 ± 0.1bc 4.0 ± 2.5ab 0.7 ± 0.2bc

8.0 ± 2.1ab 7.0 ± 1.9ab 14.8 ± 3.1a 17.0 ± 5.0a 8.0 ± 3.7ab 12.3 ± 1.7a 1.2 ± 1.0b

2.7 ± 0.4a 2.4 ± 0.6a 3.6 ± 0.3a 3.7 ± 0.5a 2.3 ± 0.8a 3.2 ± 0.2a 0.4 ± 0.4b

ANOVA T Pl TxPl

***

**

***

***

ns

ns

ns

ns

***

***

**

**

Mean values denoted by the same letter in a column do not differ significantly according to Tukey’s test (p > 0.05). In the ANOVA, T, treatment; Pl, plant (A. halimus). ** p < 0.01. *** p < 0.001.

3.2.2. Soil microbial activity Very low values of ␤-glucosidase, urease, acid phosphatase and arylsulphatase activities were found, although quite similar to those observed in soils from contaminated areas under semi-arid conditions [1,15]. Compost and pig slurry addition significantly increased these hydrolase activities, with the only exception of acid phosphatase (p < 0.01, p < 0.05 and p < 0.001 for ␤-glucosidase, urease and arylsulphatase, respectively; Fig. 1), indicating the reactivation of soil C, N, and S cycles [10]. The stimulation of enzymatic activities in degraded soils after the addition of organic amendments has been previously reported [14,15,34]. The elements supplied with these materials may activate enzyme synthesis and, simultaneously, enhance microbial development, thereby increasing enzyme concentrations in soil [35]. The presence of TEs may inhibit soil enzyme activities, due to ionic competition with enzymatic cofactors or to their interaction with substrates or specific catalytic sites [36]. However, in our study, the supply of nutrients had a greater influence on enzyme activities than TEs availability, according to the significant correlations found between these parameters (p < 0.05; Table A.1, Supporting Information). The addition of the organic amendments to the soil implies also the introduction of microorganisms present in the organic materials. Then, whether the increase of enzymatic activities is due to the activation of the soil indigenous microorganisms, or to the external input or to a combination of both remains unclear [34]. With respect to acid phosphatase, it generally increases at higher values of soil OM content [10], but no significant treatment effects were observed here (Fig. 1). This can be a consequence of the pH increase induced by the amendments [19] or the inorganic P supplied with them [6]. The same trend was observed for the overall functional activity of soil, estimated by the FDA activity [37] and the −AWCD values from Biolog EcoPlatesTM [38]. As in the case of hydrolases, FDA activity and AWCD values were higher in the organically amended soils than in the control (p < 0.001 and p < 0.01 for FDA and AWCD, respectively; Fig. 1). This fact and the positive

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140

-1

Urease

a

25

ab ab

80 60

30

ab

p<0.001

15

-1

p<0.05 ab

10 bc

c

c

b

c

b

Acid phosphatase

Arylsulphatase ab

3 2 b

a

a

FDA

b

1

b

0 0.7

AWCD

a

0.6

a

100

0.5

p<0.01

ab

80 60

a

bc bc

20

0.4

a

p<0.001

40

-1

100

120 1

4

p<0.01

150

0 140

-1

5

a

-1

200

50

mg F kg h-

6

a

p>0.05

0 7

-1

mg PNP kg h

a

mg PNP kg h

-1

250

5

b

0 300

-1

40 20

20

+

mg PNP kg h

100

a

mg N-NH4 kg h

-1

120

35

β-glucosidase

bc

0.3 0.2

ab ab

0.1

c

b

0

CT

0.0

HL-NoPlant

CM-NoPlant

PS-NoPlant

HL-Plant

CM-Plant

PS-Plant

Fig. 1. Soil enzyme activities and average well colour development (AWCD) at the end of the experiment. HL, hydrated lime; CM, compost; PS, pig slurry; CT, control.

correlations found between both parameters and BC and BN (BC FDA: r = 0.775, p < 0.001; BN -FDA: r = 0.641, p < 0.001; BC -AWCD: r = 0.733, p < 0.001; BN -AWCD: r = 0.670, p < 0.001), indicate a clear stimulation of the soil microbial communities by the organic amendments [6,39,40]. The capacity of AWCD values to show treatment effects was also revealed here, in agreement with other studies carried out in TEs contaminated soils [6,16,17], despite its limited representativeness (Biolog EcoplatesTM reflect the potential of only the heterotrophic cultivable portion of the soil bacterial community to utilise C sources) [38,41]. Kelli and Tate [11] reported that the remediation of a Zn contaminated soil with a mixture of sewage sludge and fly ash led to the recovery of similar metabolic profiles than non-affected soils, reflecting a substantial improvement of soil quality. Finally, the status of soil microbial communities was also assessed through the determination of dehydrogenase activity, basal respiration and the metabolic quotient (qCO2 ). Dehydrogenase is an intracellular enzyme related to the oxidative phosphorylation process [42], an intracellular process that occurs only in viable cells [43]. Similarly, basal respiration is an indicator of soil microbial activity closely related with OM degradation [6]. The

low values observed in the external control for both respiratory measurements and for all the other microbial parameters determined here (BC and BN , hydrolases, FDA, AWCD), which were much lower than those observed in degraded non- or less-contaminated soils [6,35,40], clearly show the unfavourable conditions for microbial growth characteristics of this soil [15,16]. However, the higher values of dehydrogenase activity and basal respiration found in compost and pig slurry (planted) treatments with respect to the control (p < 0.01; p < 0.001; Fig. 2), indicate that these materials stimulated the growth of the soil microbial communities and/or their enrichment with the microorganisms added with the amendments. In addition, a positive plant effect for dehydrogenase activity was observed (Pl: p < 0.05), in agreement with previous studies that reported a plant-induced microbial stimulation in soils under semiarid climates (including Atriplex species; [44,45]). But this plant effect was not observed for basal respiration, suggesting a different efficiency of soil microorganisms regarding organic C utilisation. Under stress conditions, microorganisms are usually less efficient and need more energy for survival, increasing the amount of CO2 evolved per substrate unit [46]. In this sense, the metabolic quotient (qCO2 ), calculated as the basal respiration to microbial biomass-C

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73

35

Dehydrogenase a

15

c b

b

b

a

5

p<0.01 a

a ab

-1 -1

10

0

qCO2

ab

400

200

bc

bc

-1

10

0 800

mg g d

15

ab

b

20

p<0.001

a

p<0.01

25

-1

mg INTF kg h

a

20

600

30 a

25

5

a

35

mg C-CO2 kg d

-1 -1

30

a

Basal resp.

b

CT HL-NoPlant HL-Plant CM-NoPlant CM-Plant PS-NoPlant PS-Plant

b

0 Fig. 2. Dehydrogenase activity, basal respiration and metabolic quotient (qCO2 ) after 2.5 years of experiment. HL, hydrated lime; CM, compost; PS, pig slurry; CT, control.

ratio, has been widely used to discern stress conditions (higher values indicate higher stress conditions) in trace elements contaminated soils [34,47], and has been often considered a valuable tool to evaluate soil health in these soils [2,12,15]. Higher respiration activity per microbial biomass unit indicates a lower metabolic efficiency, as a considerable portion of the energy resources are used to survive, resulting in less organic C incorporation into the microbial biomass [47]. Compost amended soils showed the lowest values of qCO2 , reaching values between 15% and 30% lower than in control soils (p < 0.01; Fig. 2) and therefore, the addition of this material might have alleviated the abovementioned stress conditions and improved the conditions for microbial growth and survival. These results are in agreement with those obtained by Pardo et al. [13] in an incubation experiment, and with those from other field experiments [2,12,15].

3.2.3. Functional diversity The term functional diversity refers to the relevance of different components of soil microbial communities for soil ecosystem functioning, which can be estimated by the values or ranges of values of functional characteristics that have an influence on important ecosystem processes, such as C utilisation [38]. Since C content is a key factor regulating microbial development in soils, functional aspects related to C utilisation may provide most relevant information regarding the effects of organic amendment additions on soil health. With this aim, functional diversity was estimated here using diversity indexes from the Biolog-CLPPs. Metabolic richness (S, number of utilised substrates) was higher in compost and pig slurry (with plants) treated soils than in controls (p < 0.001, Table 3), finding values similar to those observed in uncontaminated soils [17]. In addition, higher values of Shannon’s diversity index (H ) were found in all the treatments with respect to controls (p < 0.001, Table 3). This indicator reflects the functional diversity of, in our case, the heterotrophic cultivable portion of the soil bacterial community in terms of the number of species (number of utilised substrates) and their relative abundances (absorbance). In

our study, the organic amendments addition, especially compost, increased the number of utilised substrates (S), but the use of each of them (H ) did not differ between the different treatments applied to the soil. Previous studies have reported that OM incorporation to soil resulted in an increase of the functional diversity estimated by Biolog-CLPPs, showing higher S and H values than the corresponding untreated soils [39,40], also in TEs contaminated soils [6].

3.3. Ecotoxicological evaluation 3.3.1. Direct assays Plant emergence of Z. mays (maize) was not affected in any soil (LC50 could not be determined in any treatment, Table 4), but growth inhibition was observed in all the soils, except in compost treatment (no EC50 ). However, L. sativa (lettuce) was considerably more sensitive to soil toxicity than Z. mays, showing lower LC50 and EC50 values in all the soils (Table 4). A considerable inhibition of seedling emergence and (aerial) growth was observed in all soils for L. sativa, except in (planted) compost and pig slurry treated soils, where no LC50 and EC50 effects were found (Table 4). According to the toxicity classes proposed by Mantis et al. [48] based on toxic units (TU = (1/EC50 ) × 100), all the treatments led to a reduction in soil toxicity for L. sativa from “very toxic” (TU ≥ 10: CT) to “toxic” (1 ≤ TU < 10: HL-No plant, HL-Plant, CM-No Plant, P-No Plant) or “not toxic” (1 < TU: CM-Plant, P-Plant) (Table 4). Despite the fact that L. sativa is known to be a TEs contamination sensitive species [49], in the present experiment, growth inhibition did not significantly correlate with TEs availability, but it did so with CW , NW , available-K, and BC values, as well as with the soil enzyme activities (Tables A.2 and A.3, Supporting Information). Therefore, the toxicity reduction observed was most likely caused by OM and nutrients supply by the amendments, the presence of A. halimus, and the microbial community stimulation. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2014.01.003.

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Table 4 Results from the ecotoxicological tests (Z. mays, L. sativa, V. fischeri, T. platyurus) of the soils. Treatments

H. lime-no plant H. lime-plant Compost-no plant Compost-plant Pig slurry-no plant Pig slurry-plant Control

Z. mays

L. sativa

V. fischeri

T. platyurus

LC50

EC50

LC50

EC50

EC50 (t = 15 min)

EC50 (t = 30 min)

EC50

NT NT NT NT NT NT NT

85.75 80.50 NT NT 93.31 91.70 70.85

58.19 46.68 NT NT 59.86 NT 36.68

20.02 19.73 18.31 NT 13.39 NT 4.95

62.52 60.76 62.55 47.98 54.01 62.29 61.69

57.97 59.77 61.15 45.79 49.51 60.86 61.46

78.83 73.53 93.82 84.76 82.28 78.78 10.15

LC50 /EC50, contaminated soil dose (%) that caused 50% lethality or inhibition; NT, non-toxic.

3.3.2. Indirect assays A similar inhibition effect on V. fischeri’s luminescence was observed for all the treatments after 15 and 30 min of exposure (similar EC50 , Table 4). According to the toxic classes mentioned above [48], all the saturation extracts produced “toxicity” for this organism (1 ≤ TU < 10), and the amendments addition did not reduce the toxic response found in the controls. Numerous studies have reported high sensitivity of V. fischeri to TEs toxicity, and it has been considered a useful tool for the evaluation of this type of contamination [21,22]. Although, in some cases, when the soluble-TEs concentrations are low this test is not very efficient [19,20,23]. Regarding T. platyurus mortality, the application of the amendments led to a considerable reduction of soil extracts toxicity (EC50 values 9-fold greater than control soils, Table 4). The toxicity was associated with pH and TEs availability in the soil (Table A.2, Supporting Information), and for this reason differences between treatments were not observed (the effects provoked on these parameters by the amendments were quite similar). At pH values between 5 and 9 the neonates survival may not be affected [50]; but the pH increase observed from control (5.5) to treated (7.5) soils could be enough to cause a significant decrease in the toxicity of the extracts to T. platyurus. In addition, an elevated TEs sensitivity has been reported for this aquatic organism in previous toxicological studies [51–53]. Blinova [51] found EC50 values of 0.04 mg L−1 and 0.11 mg L−1 for Cu and Pb, respectively, and Heinlaan et al. [53] reported EC50 values of 0.24–0.98 mg Zn L−1 . According to the toxicity scale proposed by Bagur-Gonzalez et al. [54], based on root elongation (R) values, the water extract of control soil was “moderately toxic” (R = 50–75%) and “very toxic” (R = 25–50%) for L. sativa and L. sativum, respectively (Table 5). The amendments additions led to a reduction in this toxicity to “slightly” (R > 75%) and “not toxic” (R = 100%). In general, L. sativum was more sensitive than L. sativa, and the toxicity decrease was less marked in the former, as a consequence of its higher sensitivity to TEs [55]. But the soil toxicity observed could be partially due to its elevated salinity, as R and GI values of L. sativa slightly increased when a solution with the same EC as the soil extract was used as blank solution instead of distilled water. L. sativa, is considered moderately sensitive to salinity [56,57] and relatively sensitive to TEs [58]. Therefore, the use of saline solutions allows disregarding EC effects and discerning TEs toxicity. The lower toxicity of soil extracts provoked by compost and pig slurry addition with respect to hydrated lime (for both species) was related to nutrients supply (positive correlations between R and GI with CW , NW , available-P and -K for L. sativum), and especially to the stimulation of the soil microbial communities (correlations between R and IG with FDA and AWCD, Tables A.2 and A.3, Supporting Information). This fact highlights the importance of chemical and biological fertility of soil in the remediation of TEs contaminated soils.

3.4. Integration of physicochemical and biological parameters A Principal Component Analysis (PCA) was carried out to relate soil physicochemical with biological properties, in an attempt to obtain a more integrated evaluation of soil health [7,8], and therefore, to better assess the effectiveness of the remediation treatments [6]. The PCA resulted in two principal components (PCs) that explained 70.3% of total variance (Fig. 3a). In PC1 (51.5% of the variance) most of the microbial parameters were positively correlated with CW , NW and available-K, and negatively with qCO2 . This information indicates that the addition of essential nutrients was crucial to stimulate the development, activity and complexity of soil microbial communities (autochthonous and/or immigrant) and to reduce their stress, allowing in turn the re-activation of the major nutrient cycles. In the PC2 (18.8% of the variance) soil pH was positively correlated with extractable-As and acid phosphatase activity, and negatively with CaCl2 -extractable TEs, showing that soil pH was the key factor for the solubility of TEs and the distribution of acid phosphatase activity. The spatial distribution of the treatments (despite showing some heterogeneity) allowed the identification of certain tendencies resulting from amendments application and A. halimus presence. Thus, all treatments provoked a similar reduction of TEs availability through a pH increase, but compost application, independently of the plant presence, and the combination of pig

Table 5 Root elongation (R) and germination index (GI) of L. sativum and L. sativa (% ± SE). Treatments

L. sativum H. lime-no plant H. lime-plant Compost-no plant Compost-plant Pig slurry-no plant Pig slurry-plant Control ANOVA L. sativa H. lime-no plant H. lime-plant Compost-no plant Compost-plant Pig slurry-no plant Pig slurry-plant Control ANOVA

H2 O as blank

CaCl2 as blank

R

IG

R

IG

51.8 ± 0.3 d 51.9 ± 1.4 d 64.0 ± 0.8 c 71.8 ± 2.1 ab 67.9 ± 0.8 bc 72.6 ± 1.1 a 47.0 ± 0.1 d

45.4 ± 0.2 c 43.9 ± 2.0 c 58.7 ± 2.6 b 69.6 ± 3.1 a 62.3 ± 0.7 ab 64.4 ± 4.3 ab 41.3 ± 0.1 c

54.8 ± 0.3 d 54.9 ± 1.5 d 67.7 ± 0.8 c 76.0 ± 2.3 ab 71.8 ± 0.8 bc 76.8 ± 1.1 a 49.8 ± 0.1 d

47.1 ± 0.3 c 45.6 ± 2 c 60.9 ± 2.7 b 72.2 ± 3.2 a 64.7 ± 0.7 ab 66.8 ± 4.4 ab 42.8 ± 0.1 c

***

***

***

***

67.3 ± 4.8 cd 69.1 ± 3.9 bcd 92.9 ± 1 abc 96.9 ± 2.4 abc 96.2 ± 6.4 ab 105.8 ± 14.9 a 51.2 ± 10.8 d

60.5 ± 2.4 bc 64.9 ± 3.7 bc 92.0 ± 2.3 ab 95.9 ± 3.8 a 95.2 ± 5 a 103.6 ± 14.6 a 47.3 ± 11.4 c

73.5 ± 5.2 bc 75.4 ± 4.3 bc 101.4 ± 1.1 ab 105.8 ± 2.7 a 105.1 ± 7 a 115.5 ± 16.3 a 55.9 ± 11.8 c

66.1 ± 2.6 bc 70.9 ± 4 bc 100.4 ± 2.5 ab 104.7 ± 4.1 a 104.0 ± 5.4 a 113.2 ± 16 a 51.6 ± 12.4 c

**

**

**

**

Mean values denoted by the same letter in a column do not differ significantly according to Tukey’s test (p > 0.05). ** p < 0.01. *** p < 0.001.

T. Pardo et al. / Journal of Hazardous Materials 268 (2014) 68–76

75

2 1.0

pH Phosph

0.0

1

As Av-K AWCD Ure S Dehy FDA Av-P NW BN B CW C

qCO2 EC

0

-1 HL-NoPlant HL-Plant CM-NoPlant CM-Plant PS-NoPlant PS-Plant CT

-0.5 Pb Cd

-1.0

Zn

Mn

CP2

CP2

0.5

b

a

-2

-3

-4 -1.0

-0.5

0.0

0.5

1.0 -2

CP1

-1

0

1 CP1

2

3

4

Fig. 3. Principal component analysis of physicochemical and biological parameters. HL, hydrated lime; CM, compost; PS, pig slurry; CT, control.

slurry with A. halimus were the most effective treatments regarding the stimulation of soil microbial communities and a concomitant improvement of soil health.

4. Conclusions The studied mine spoil soil showed significant toxicity due to its high salinity, slight to moderate acidic pH and TEs solubility, but the application of the amendments led to a reduction of potential associated risks. The toxicity decrease, together with the supply of essential nutrients by pig slurry and compost addition, promoted the development of a stable vegetation cover that simultaneously supposed an extra input of nutrients. This enlargement of soil nutrients stimulated soil microbial communities (autochthonous and/or immigrant), reducing their stress and increasing their growth, activity and functional diversity. This meant an improvement of the habitat function of the soil ecosystem, and the reactivation of the biogeochemical cycles of essential nutrients in the soil. Therefore, under semi-arid conditions, the use of compost and pig slurry, in combination with A. halimus, seems to be an effective phytostabilisation strategy to improve soil health of TEs-contaminated soils. Larger scale application of this procedure in the Sierra Minera of La Unión-Cartagena or in other similar areas with similar edaphoclimatic conditions seems a feasible and environmentally–friendly soil restoration option.

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