Sewage sludge application can induce changes in antioxidant status of nodulated alfalfa plants

ARTICLE IN PRESS Ecotoxicology and Environmental Safety 73 (2010) 436–442

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Sewage sludge application can induce changes in antioxidant status of nodulated alfalfa plants M. Carmen Antolı´n n, Iara Muro, Manuel Sa´nchez-Dı´az ´n Biologı´a Vegetal (Unidad Asociada al CSIC, EEAD, Zaragoza), Facultades de Ciencias y Farmacia, Universidad de Navarra, Dpto. Biologı´a Vegetal, Seccio C/Irunlarrea 1, 31008 Pamplona, Spain

a r t i c l e in f o

a b s t r a c t

Article history: Received 9 February 2009 Received in revised form 16 June 2009 Accepted 30 August 2009 Available online 2 December 2009

A greenhouse experiment was conducted to investigate the oxidative stress produced by sewage sludge addition on nodulated alfalfa (Medicago sativa L. cv. Arago´n) plants. Two types of sludge were incorporated into substrate: anaerobic mesophilic digested (AM) and autothermal thermophilic aerobic digested (ATAD) sludge. Pots without sludge but with inoculated plants were used as control treatment for comparison. Results showed that sludge amended plants had increased tissue accumulation of heavy metals that induced oxidative stress. This is characterized by induction of the antioxidant enzymatic activities and alterations in the redox state of ascorbate. ATAD sludge application produced a reduction in nodulation, increased nodule antioxidant enzyme activities and decreased ascorbate/ dehydroascorbate ratio. As a consequence, nodules of ATAD treatment suffered from oxidative damages as evidenced by high malondialdehyde levels. By contrast, AM application enhanced plant growth and no deleterious effects on nodulation were found. Nodules developed in AM sludge had increased antioxidant enzyme activities, ascorbate/dehydroascorbate ratio and improved capacity for thiol synthesis. Results clearly showed that nodulated alfalfa performed better in AM than in ATAD sludge and suggest that differential response appears to be mediated by plant ability to thiol synthesis and to maintenance of a more equilibrated antioxidant status. & 2009 Elsevier Inc. All rights reserved.

Keywords: Autothermal thermophilic aerobic digestion (ATAD) Heavy metals Nitrogenase activity Oxidative stress Sewage sludge

1. Introduction The progressive implementation in 2005 of the Directives 91/271/EEC and 98/15/EEC concerning urban wastewater treatment has increased the number of wastewater treatment plants operating in the EU and consequently the quantities of sewage sludge requiring disposal. Sewage sludge are organic C-rich materials produced during wastewater treatment and represent a source of organic matter, nitrogen, phosphorus and other nutrients, which, if properly managed, can be used to improve organic fertility in intensively cropped degraded soils of Mediterranean climate zone (Garcı´a et al., 2000; Garcı´a-Gil et al., 2004; Ferna´ndez et al., 2009). In Spain, the abundance of carbonate-rich soils, with their low organic matter content, favours the application of sewage sludge as an organic amendment and nutrient supply to soil with a relatively small risk of pollution (Navas et al. (1998); Antolı´n et al., 2005). However, the presence of organic contaminants and heavy metals, commonly present in organic waste, must always be controlled before their application due to their dangerous effects over the ecosystem (Carbonell et al.,

n

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0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2009.08.022

2009). Restrictions on agricultural use of sewage sludge in Europe were mainly based on metal concentrations. In the European Union (EU) Directive 86/278/EEC of June 1986, limit values of the total amount of several heavy metals were established for land application of sludge. In 2000, the EU published the third draft of a future sludge directive where more restricted concentration limit values for heavy metals are fixed. Oxidative stress occurs when there is a serious imbalance in any cell compartment between the production of reactive oxygen species (ROS) and antioxidant defense, leading to dramatic physiological challenges (Polle, 1997). Soil accumulation of heavy metals produced after successive sludge applications can induce oxidative stress in plants and microorganisms (Grata~ o et al., 2005; Sinha et al., 2005; Reddy et al., 2005). Indeed, some studies have reported detrimental effects on plant–microbe symbiotic interactions involving rhizobial bacteria resulting from sludge application (McGrath et al., 1988; 1995; Gibbs et al., 2006). Cells possess both enzymatic and non-enzymatic defense systems to maintain the cellular redox state and to mitigate the damage caused by oxidative stress (reviewed in Apel and Hirt, 2004). Legume nodules contain a variety of enzymes and metabolites (antioxidants) to prevent the formation of potentially toxic concentrations of ROS and subsequent oxidative stress. Non-enzymatic defense typically consist of small molecules, which act as radical scavengers, being

ARTICLE IN PRESS M. Carmen Antolı´n et al. / Ecotoxicology and Environmental Safety 73 (2010) 436–442

oxidized by ROS and thereby removing them from solution. These molecules include the major cellular redox buffers ascorbate (ASC) and glutathione, as well as tocopherol, flavonoids, alkaloids and carotenoids. In legume nodules, the most abundant watersoluble antioxidants are ASC, glutathione and homoglutathione (Matamoros et al., 2003, 2006). Cellular antioxidative defenses also include several enzymes, which are capable of removing ROS and their products. These enzymes are superoxide dismutase, catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). APX and GR are important components of the ASC–glutathione cycle, which is also involved in the removal of the hydrogen peroxide (H2O2) in the cell (reviewed in Grata~ o et al., 2005). Sewage sludge properties depend on the type of wastewater, the technology used and on the time of the year. At present, attention is being focused on the characteristics of the end product, which originate in the wastewater plant, particularly, when the sludge is used for agricultural land (Singh and Agrawal, 2008). Anaerobic digestion of sludge involves biologically stabilizing sludge in a closed vessel to reduce the organic content, mass, odor and pathogens under mesophilic temperatures (35 1C). Aerobic digestion utilizes oxygen or air to biologically stabilize sludge. The organic matter is converted to carbon dioxide, water and nitrogen, and pathogens and odors are reduced. Autothermal thermophilic aerobic digestion (ATAD) is a process where the higher temperatures result in effective pathogen destruction (Riley and Foster, 2001; Juteau, 2006; Layden et al., 2007). ATAD is characterized by high substrate removal rates, low mass of residual sludge and reduction of pathogens in the remaining sludge (Wiesmann and Libra, 1999). Other environmental benefits associated with ATAD technology are the reduction of sludge volume and methane emissions, thus land application of ATADtreated sludge could be a feasible disposal method. Despite apparent benefits of this process, soil application of sludge obtained from ATAD technology is still not well understood. Therefore, the aim of this study was to compare the response of antioxidant system of nodulated alfalfa plants to application of a sludge obtained from ATAD technology and a conventional sludge (anaerobic mesophilic). Specifically we sought to identify the extent of antioxidative protection in sludge-treated nodules.

437

Table 1 Sewage sludge properties. Element/population

Sludge AM

Sludge ATAD

Dry mass (%) pH EC (mS cm  1) TOC (%) COD total (mg l  1) Nkjeldhal (%) Ptotal (%) Ktotal (%) C/N NH4+ (mg l  1) Catotal (%) Mgtotal (%) Natotal (%) Fetotal (%) Cdtotal (mg kg  1) Crtotal (mg kg  1) Cutotal (mg kg  1) Mntotal (mg kg  1) Nitotal (mg kg  1) Pbtotal (mg kg  1) Zntotal (mg kg  1) Escherichia coli (cfu/g DM) Salmonella spp. Clostridium perfringens (cfu/100 g DM)

2.90 7.6 9.91 30.0 22,000 9.5 3.0 0.9 3.9 1,300 5.4 0.7 0.5 0.9 o3 161 286 171 49 81 990 60 ND 38,000

2.83 8.5 7.97 33.0 31,600 8.6 3.4 0.9 3.1 1,300 4.3 0.8 0.6 0.7 o3 128 275 169 41 50 940 o 10 ND 600

EC Electric conductivity, TOC total organic carbon, COD chemical oxygen demand, DM dry mass. remove any trace of chemical that could interfere in seed germination and placed in Petri dishes to germinate. Petri dishes were watered daily till seed germination using sterile distilled water. After five days, six seedlings of alfalfa were transplanted into each pot. During the first month, plants were inoculated three times with Sinorhizobium melioti strain 102F34 maintained on yeast extract mannitol agar (Ba´scones et al., 2000), which was classified as a Hup  strain (Brito et al., 2005). Plants were grown in a glasshouse at 25 1C/15 1C and 50%/70% RH (day/night). The photoperiod was 14 h under natural daylight, supplemented with high pressure sodium lamps (SON-T Agro Phillips, Eindhoven, The Netherlands), which provided a minimum photosynthetic photon flux of about 400 mmol m  2 s  1 at the upper level of canopy. Plants were watered twice a week with Evans N-free nutrient solution (Evans, 1974) alternating with deionized water to avoid salt accumulation under pots. Plants were harvested 60 days after emergence. Five plants from different pots were taken for different analysis.

2.3. Nodule determinations 2. Materials and methods 2.1. Sludges The sewage sludge was collected at the wastewater plant of Tudela (Navarra, Spain), which processes domestic wastewater amounting to 38,969 person equivalents per year. The raw sludge was treated with two different processes: (1) anaerobic digestion carried out at 35 1C for a period of 20 days, and which is referred to as anaerobic mesophilic sludge (AM); and (2) autothermal termophilic aerobic digestion (ATAD) carried out at 55–60 1C for 7 days. Table 1 shows the main properties of sludges. Heavy metals were below the limits permitted by EU Directive 86/278/EEC. 2.2. Experimental design Two hundred grams of a mixture of perlite and vermiculite (2:1, v/v) was packed into 25  10 cm pots (2.0 dm3 volume). The two sludges were incorporated into substrate at different rates, from 1%, 5% and 10% (w/w), which were equivalent to approximately 3, 15 and 30 t dry matter ha  1, respectively. These rates fall within the ranges commonly applied in semiarid Mediterranean soils (Antolı´n et al., 2005; Ferna´ndez et al., 2009, Tarraso´n et al., 2009). Ten replications per treatment were prepared. The sludges were added to the substrate 30 days before planting, in order to allow the chemical degradation, biodegradation and volatilization of toxic compound in sludges reach equilibrium in substrate and thus reduce the risk of phytotoxicity. Pots without sludge but with inoculated plants were used as control treatments for comparison. Seeds from alfalfa (Medicago sativa cv. Arago´n) were surface disinfected in a 0.1% (w/v) HgCl2 solution for 10 min, washed five times with sterile water to

Apparent nitrogenase activity (ANA) was measured as H2 evolution on the intact plants with root systems sealed in the growth pots and housed inside a chamber in an open flow-through system under N2:O2 (79:21%) according to Witty and Minchin (1998), using an electrochemical H2 sensor (Qubit System Inc., Canada) as described by Gonza´lez et al. (2001), but a flow rate of 460 ml min  1. The detector was calibrated with high purity gases (Praxair, Madrid, Spain) using a gas mixer (Air Liquid, Madrid, Spain) flowing at the same rate as the sampling system. After measurement of nitrogenase activity, roots were carefully washed and nodules were detached, counted, weighed and stored at  80 1C until analysis. Leghemoglobin was extracted three times, each with 2 ml of Drabkin’s solution obtained by adding 52 mg of potassium cyanide, 198 mg of potassium ferricyanide and 1 g of sodium bicarbonate to 1000 ml of distilled water (Becana et al., 1986). Nodule samples were centrifuged at 30,000g at 4 1C for 15 min and the supernatant was used for leghemoglobin and nodule soluble protein determinations. Leghemoglobin was measured by reading the absorbance using a spectrophotometer at 540 nm (LaRue and Child, 1979). Total soluble proteins were quantified by grinding and filtering 100 mg of fresh matter frozen tissue in a cold mortar using an extraction buffer containing 50 mM K-phosphate (pH 7.5). Extract was filtered and centrifuged at 28,710g at 4 1C for 15 min. The supernatant was used for protein determination. Total proteins were measured with the protein dye-binding method using bovine serum albumin as a standard (Bradford, 1976).

2.4. Markers of oxidative stress Frozen plant material was used to determine hydrogen peroxide (H2O2) and oxidative damage of lipids. The H2O2 measurements were essentially made as described by Patterson et al. (1984) with some modifications (Erice et al., 2007).

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Three hundred milligrams of the nodules were homogenized in a cold mortar with 5 ml 5% trichloroacetic acid (TCA) containing 0.1 g activated charcoal and 0.1% polyvinylpolypyrrolidone (PVPP). The homogenate was filtered and centrifuged at 18,000g at 4 1C for 10 min. The supernatant was filtered through a Millipore filter (0.45 mm) and used for the assay. A 200-mL aliquot was brought to 2 ml with 100 mM potassium phosphate buffer (pH 8.4) and 1 ml of a colorimetric reagent was added. This reagent was prepared daily by mixing 1:1 (v/v) 0.6 mM potassium titanium oxalate and 0.6 mM 4-2 (2-pyridylazo) resorcinol (disodium salt). After incubating the sample solution at 60 1C for 45 min, the absorbance was measured at 508 nm. Blanks were made by replacing leaf extract with 5% TCA. Lipid peroxidation was estimated as the content of malondialdehyde (MDA) formed from decomposition of lipid peroxides at acidic pH after reaction with thiobarbituric acid (TBA) (Zornoza et al., 2002). Plant material (100 mg of fresh matter) was extracted in 2.0 ml of TCA–TBA–HCl reagent (15% (w/v) TCA, 0.37% (w/v) TBA and 0.25 mM HCl). The extract was heated in a bath at 90 1C for 30 min. After cooling, the flocculent precipitate was removed by centrifugation at 11,000g at 4 1C for 10 min. Absorbance of the supernatant was measured at 535 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm.

2.5. Antioxidant metabolites Frozen plant material was used to determine antioxidant metabolites. Total thiols were assayed shaking 100 mg of fresh matter of plant material with 0.4 ml of NaBH4 (1 mg ml  1) dissolved in NaOH 1 M and 0.2 ml of deionized water. After centrifugation at 11,000g at 4 1C for 10 min, 0.5 ml of supernatant was added to 0.5 ml of 5,50 -dithiobis(2-nitrobenzoic acid) dissolved in a buffer (0.5 M K-phosphate pH 7.2). Absorbance was measured at 410 nm (Jocelyn, 1987). Ascorbate (ASC) and dehydroascorbate (DHA) content were analyzed only in nodules and were assayed photometrically by reduction of 2,6-dichlorophenolindophenol (DCPIP) following published protocol (Leipner et al., 1997). Nodules were homogenized in liquid nitrogen in the presence of 1 g NaCl and extracted in 5 ml ice-cold 2% (w/v) metaphosphoric acid. The homogenate was filtered. An aliquot of 0.3 ml was mixed with 0.2 ml 45% (w/v) K2HPO4 and 0.1 ml 0.1% (w/v) homocysteine to reduce DHA to ASC and determine the total ASC pool (ASC +DHA). For the determination of ASC, the homocysteine solution was replaced by the same volume of water. After 15 min incubation at 25 1C, 1 ml of citrate–phosphate buffer 2 M (pH 2–3) and 1 ml 0.003% (w/v) DCPIP were added. The absorbance at 524 nm was measured immediately using a spectrophotometer. The content of ASC was calculated by reference to a standard curve. The amount of DHA resulted from the subtraction of the total ASC pool (ASC+ DHA) and ASC.

2.6. Antioxidant enzyme activities Two hundred milligrams of the plant material were homogenized in 5 ml 100 mM phosphate buffer (pH 7.0) containing 0.1 mM DTPA and 50 mg PVPP with a pestle and mortar. The homogenate was filtered and centrifuged at 38,000g at 4 1C for 10 min. The supernatant was collected and assayed for antioxidant enzyme activities as described in Aroca et al. (2001). Ascorbate peroxidase (APX, EC 1.11.1.7) was assayed by the ASC oxidation method at 290 nm for 3 min after adding 100 mL of extract. Glutathione reductase (GR, EC 1.6.4.2) activity was determined by NADPH2 oxidation method at 340 nm for 3 min after adding 200 mL of extract. Catalase (CAT, EC 1.11.1.6) activity was determined according to Aebi (1974) by measuring H2O2 decomposition at 260 nm for 30 s after adding 200 mL of extract. All enzymatic activities were measured at 25 1C.

2.7. Other determinations

2.8. Statistical analysis Data were submitted to a two-factor analysis of variance (ANOVA). Variance was related to the main treatments (presence of sludge and organ) and to interaction between them. Means7 standard errors (S.E.) were calculated and, when F ratio was significant, least significant differences were evaluated by a Tukey’s t-test, as found in the Statistical Package for the Social Sciences (SPSS) (SPSS Inc., Chicago, USA) version 15.0 programs for Windows XP. All values shown in the figures are means7 SE.

3. Results 3.1. Soil properties and heavy metals The main properties of perlite/vermiculite mixtures assessed at the end of experimental period are shown in Table 2. The addition of AM sludge to substrate decreased pH, increased electric conductivity (EC) and concentrations of N, P and K. ATAD sludge amendment increased soil concentrations of N, P and K. Application of AM or ATAD sludge resulted in an increase soil DTPA-extractable heavy metals compared with control treatment, being availability of Cr, Cu, Mn and Ni higher in AM than in ATAD treatments. Heavy metals in the dry mass (DM) of alfalfa plants are summarized in Table 3. Two-factorial ANOVA showed significant interactions between sludge and organ for most of elements analyzed because leaves and roots responded in a different way to sludge application. In leaves, sludge application increased concentrations of Cu, Mn and Zn in AM and Mn and Zn in ATAD-treated plants. However, in roots, application of AM or ATAD sludge produced significant accumulation of all heavy metals, especially in AM-treated plants. 3.2. Plant growth and nodule activity The addition of increasing rates of sludge stimulated production of alfalfa DM only in AM-treated plants with respect to untreated plants (Fig. 1). By contrast, ATAD-treated plants achieved similar sustained growth in all rates assayed. When compared both types of sludge at rate of 10% (w/w, which were equivalent to approximately 30 t dry matter ha  1) it was evident that plants grown in ATAD sludge produced lower nodule DM (Fig. 1) and nodule number (Table 4) than those grown in AM, and that, in turn, was reflected in low plant DM (Fig. 1). Since significant differences between AM and ATAD-treated plants were detected at 10% (w/w) of sludge addition, the rest of parameters Table 2 Some substrate properties in the mixture of perlite and vermiculite (2:1, v/v) amended with 10% (w/w) of anaerobic mesophilic (AM) or ATAD sludge and untreated (control). Parameter

Plants organs were weighed separately after being separated into leaves, stems, roots and nodules. Dry mass (DM) was obtained by drying samples in an oven at 80 1C until a constant mass was obtained. Nitrogen (N) content was determined in dried leaf tissue by using the Kjeldahl method and crude protein concentration was estimated as N  6.25 (Padmore, 1990). Phosphorus (P) was extracted with NaHCO3 (Olsen et al., 1954). Potassium (K) was extracted with ammonium acetate and analyzed by flame spectrometry. Soil pH was measured in an aqueous solution (1:10 w/v) and electrical conductivity (EC) was measured in 1:10 water extract. Heavy metal concentrations in substrate and plants were determined following nitric–perchloric acid digestion. The ‘‘plant available’’ metal concentrations in substrate were determined after extraction with 0.005 M DTPA (Lindsay and Norvell, 1978). All plant and substrate material digests were analyzed for Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn using inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7500, Agilent Technologies, Spain). Quality control was assured by the use of certified reference materials SRM 1575a (pine needles) and BCR 100 (beech leaves) for plants and CMI 7003 (silty clay loam soil) for soils, procedural blanks and duplicates of the analysis.

pH EC (mS cm  1) Nkjeldhal (g 100  1) Polsen (mg kg  1) Kavailable (mg g  1) Feavailable (mg kg  1) Cdavailable (mg kg  1) Cravailable (mg kg  1) Cuavailable (mg kg  1) Mnavailable (mg kg  1) Niavailable (mg kg  1) Pbavailable (mg kg  1) Znavailable (mg kg  1)

Control

AM

ATAD

7.2a 1.12b 0.03c 54.6c 217.4c 6.23b o 0.001b 0.007c 0.95c 3.62c 0.027c 0.03c 0.22b

6.7b 1.76a 0.42b 779.5a 1350.5a 18.15a 0.036a 0.129a 9.62a 8.02a 1.515a 0.54b 29.48a

7.0a 1.21b 0.53a 651.0b 842.3b 15.42a 0.037a 0.081b 7.10b 6.79b 0.790b 0.77a 27.75a

EC Electric conductivity. Within each parameter, means followed by a common letter are not significantly different (P o 0.05) according to a Tukey’s test. Values are means (n= 5).

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Table 3 Heavy metal concentrations in alfalfa grown in 10% (w/w) of anaerobic mesophilic (AM) or ATAD sludge and untreated soils (control). Treatment

Cd (mg kg  1)

Cr (mg kg  1)

Cu (mg kg  1)

Fe (mg kg  1)

Mn (mg kg  1)

Ni (mg kg  1)

Pb (mg kg  1)

Zn (mg kg  1)

Leaves

Control AM ATAD

0.04b 0.05b 0.03b

0.39d 1.79d 1.42d

8.41d 16.26c 14.01cd

216.6b 331.6b 505.6b

52.92d 120.61c 158.06bc

0.79d 3.51d 1.98d

0.10b 0.16b 0.14b

14.11c 35.28b 40.42b

Roots

Control AM ATAD

0.10b 0.25a 0.32a

14.80c 55.98a 38.33b

19.41c 45.83a 31.43b

2520.3b 11988.8a 12697.0a

56.76d 249.28a 194.56ab

11.68c 39.41a 20.00b

0.24b 1.76a 1.90a

12.89c 65.65a 78.38a

F = 15.40nnn F = 139.23nnn F = 19.43nnn

F= 22.83nnn F= 185.44nnn F= 19.89nnn

F = 54.20nnn F = 206.91nnn F = 16.42nnn

F =24.91nnn F =168.57nnn F =22.87nnn

F =39.25nnn F =17.65nnn F =7.77nnn

F= 38.17nnn F= 220.33nnn F= 26.13nnn

F =28.39nnn F =129.03nnn F =25.09nnn

F= 91.217nnn F= 57.815nnn F= 16.622nnn

Presence of sludge Plant organ Interaction

Within each element, means followed by a common letter are not significantly different (Po 0.05) according to a Tukey’s test. Values are means (n= 5). nnn

Significance at 0.001 probability level.

Nodule DM (g plant-1)

Plant DM (g plant-1)

2.5 2.0 1.5

AM ATAD

Table 4 Main plant characteristics and nodule activity in alfalfa plants grown in 10% (w/w) of anaerobic mesophilic (AM) or ATAD sludge and untreated soils (control).

a ab ab

ab

ab b

b

1.0 0.5

0.03

a

a a

a

a

0.02

a

b

0.01 0

Measurement

Control

AM

ATAD

Forage quality Crude protein (g kg  1) P (g kg  1) K (g kg  1)

282.75b 5.64b 29.33a

336.88a 12.29a 35.90a

322.81a 11.02a 31.11a

Nodule characteristics Nodulation (nodule plant  1) ANA (mmol g-1 NDM min  1) ANA (mmol plant  1 min  1) Total soluble proteins (mg g  1 NDM) Leghemoglobin (mg g  1 NDM)

36.6a 1.72ab 0.043a 161.93a 32.64a

37.6a 0.97b 0.031ab 75.77b 23.25a

18.6b 2.11a 0.025b 80.44b 21.33a

NDM: nodule dry matter. Within each parameter, means followed by a common letter are not significantly different (P o 0.05) according to a Tukey’s test. Values are means (n= 5).

Control

1 5 Sewage sludge (%)

10

Fig. 1. Plant and nodule dry mass of alfalfa plants grown in soils treated with increasing rate of sewage sludge obtained from different processing treatments: anaerobic mesophilic (AM) or autothermal thermophilic aerobic (ATAD) and untreated (control). Values represent means (n= 5); bars indicate standard error (S.E.) of the mean. Different letters indicate significant differences (p r 0.05) between rates and treatments according to a Tukeys test.

were analyzed only in plants grown at this rate. Results of Table 4 also showed that application of sludge produced, firstly, a significant increase of forage quality as indicated by the amount of crude protein, and secondly, higher concentration of P. Nodules of ATAD treatment showed higher apparent nitrogenase activity (ANA) than those AM-treated plants (Table 4). However, on a plant basis, ANA was significantly lower in ATAD than in AM-treated plants due to reduced nodulation of the former (Fig. 1). Moreover, there was a clear effect of sludge application on nodule soluble proteins. Thus, nodules of untreated plants displayed the highest concentration of nodule proteins, and the two sludge types produced a significant reduction in this parameter. By contrast, sludge addition did not induce any significant variation in nodule leghemoglobin content.

3.3. Oxidative stress Oxidative damage of lipids was estimated by measuring malondialdehyde (MDA), which is one of the decomposition

Table 5 Markers of oxidative stress in different plant organs of alfalfa grown in 10% (w/w) of anaerobic mesophilic (AM) or ATAD sludge and untreated soils (control). Treatment

MDA (nmol g  1 DM)

H2O2 (mmol g  1 DM)

Leaves

Control AM ATAD

153.91bc 119.60bc 138.02bc

2.31abc 2.21abc 2.29abc

Roots

Control AM ATAD

87.20c 177.06b 178.73b

3.69a 3.47ab 2.01bc

Nodules

Control AM ATAD

179.79b 148.61bc 300.88a

2.83ab 1.10c 3.10ab

F= 7.26nnn F= 14.93nnn F= 8.78nnn

F= 3.27 nsa F= 4.92nn F= 7.14nnn

Presence of sludge Plant organ Interaction

DM: dry matter. Within each parameter, means followed by a common letter are not significantly different (Po 0.05) according to a Tukey’s test. Values are means (n= 5). a

Not significant. Significance at 0.001 probability level. Significance at 0.01 probability level.

nnn nn

products of polyunsaturated fatty acids of biomembranes. There was a clear effect of sludge application on MDA levels, showing that AM or ATAD sludge caused significant increases in MDA in roots, but there were no significant changes in leaves (Table 5). In

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nodules, application of ATAD resulted in a significant increase in MDA concentration. Moreover, there were little differences in the amount of hydrogen peroxide (H2O2) in leaves, but in roots, ATAD-treated plants exhibited lower H2O2 than in untreated plants. In nodules, AM-treated plants had lower H2O2 accumulation than those other treatments. These differential patterns were emphasized by two-way ANOVA because there were significant interactions between organs and presence of sludge for both parameters.

3.4. Antioxidant status and enzyme activity The activities of some antioxidant enzymes are shown in Table 6. Two-way ANOVA revealed significant interactions between organs affected and sludge addition in all enzymes tested. Thus, in leaves and roots, sludge application had little effect on the activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). By contrast, in nodules, all enzyme antioxidant activities analyzed were strongly stimulated by the presence of AM or ATAD sludge. Moreover, APX activity was higher in nodules developed in ATAD sludge than in those treated with AM. This coincided with a significant increase in dehydroascorbate (DHA) in ATAD nodules (Table 7). Measurements of ascorbate (ASC) pool indicated that the ASC+ DHA (total ASC) decreased in nodules treated with AM or ATAD sludge in comparison with untreated controls. The ASC redox state (estimated as ASC/DHA) was altered by the presence of both types of sludges in a different way. In ATAD-treated nodules, ASC was strongly reduced whereas DHA accumulated. By contrast in AM-treated nodules, ASC was predominant over DHA. Finally, nodule thiol pool was also significantly enhanced in the presence of AM sludge (Table 7).

4. Discussion It is well understood that the addition of sewage sludge improves soil fertility due to increased availability of N and P. These changes can result in improved plant growth (Pascual et al., 2004; Antolı´n et al., 2005), which was also evident in this study only when high rates of AM sludge were applied (Fig. 1). ATAD sludge can also increase growth and yield of pepper plants (Pascual et al., 2008), but the present study showed that these positive effects of ATAD were not as obvious in rhizobia–legume associations. Some authors stated that positive response of crops to sludge application is not a general phenomenon, and could depend, at least in part, on the soil type, the sludge applied and/or the technology used for processing raw material, which in turn, could have a decisive impact on the agronomic quality of the end product (Richards et al., 2000; Kim et al., 2007; Singh and Agrawal, 2008). In alfalfa, the presence of AM or ATAD sludge favoured high leaf crude protein (high forage quality) and leaf P concentration compared to untreated plants (Table 4). Sludge addition can produce some modifications in soil, such as declines in pH, increases in EC and increases in heavy metal content and its availability for crops (Navas et al. (1998); Hao and Chang, 2003; Pascual et al., 2004; Kim et al., 2007). All these effects became present in substrate amended with AM sludge, probably due to the accumulation of soluble salts (i.e., phosphates) in the substrate (Table 2). In ATAD-treated pots, only increases in availability of heavy metals were observed (Table 2). Both types of sludges meet the standards of heavy metal contents, regardless of the process by which they were obtained (Table 1), but substrate amended with AM exhibited higher amounts of available heavy metals than ATADtreated soil that could have resulted, at least in part, from decline in pH of the former (Table 2) (Richards et al., 2000; Kim et al., 2007). On the other hand, it is necessary to take into account that, according to the process used with wastewaters, the resulting sludges could

Table 6 Antioxidant activities in different plant organs of alfalfa grown in 10% (w/w) of anaerobic mesophilic (AM) or ATAD sludge and untreated soils (control). Treatment

APX (mmol g  1 DM s  1)

GR (mmol g  1 DM s  1)

CAT (mmol g  1 DM s  1)

Leaves

Control AM ATAD

0.231c 0.034d 0.114cd

0.257a 0.083bc 0.156b

7.36b 6.87bc 8.93b

Roots

Control AM ATAD

0.054d 0.022d 0.017d

0.049c 0.025c 0.034c

5.39bc 1.47c 4.11bc

Nodules

Control AM ATAD

0.187c 0.658b 0.938a

0.067c 0.264a 0.237a

13.56b 63.62a 47.66a

Presence of sludge Plant organ Interaction

F =13.24nnn F =120.38nnn F =27.40nnn

F= 0.73nnn F= 44.53nnn F= 20.48nnn

F =6.31nnn F =64.61nnn F =17.50nnn

DM: dry matter. Within each parameter, means followed by a common letter are not significantly different (P o0.05) according to a Tukey’s test. Values are means (n= 5). nnn

Significance at 0.001 probability level.

Table 7 Antioxidant metabolites in nodules of alfalfa grown in 10% (w/w) of anaerobic mesophilic (AM) or ATAD sludge and untreated soils (control). Treatment

Thiols (mmol g  1 DM)

ASC (mmol g  1 DM)

DHA (mmol g  1 DM)

ASC/DHA

ASC+ DHA (mmol g  1 DM)

Control AM ATAD

11.04b 26.64a 15.74b

39.63a 47.30a 2.07b

29.98b 6.63c 42.63a

1.20b 8.58a 0.05c

69.61a 53.93b 44.69b

DM: dry matter. Within each parameter, means followed by a common letter are not significantly different (P o0.05) according to a Tukey’s test. Values are means (n= 5).

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have different chemical characteristics. For instance, Merrington et al. (2003) have reported that heavy metals associated with organic matter differs in aerobically and anaerobically digested sludges, which in turn, could affect the content of heavy metals in plants (Seyhan and Erdincler, 2003; Warman and Termeer, 2005). Moreover, it was reported that metal availability is dependent on the form of organic matter present in the sludge (Kim et al., 2007). As expected, application of AM or ATAD sludge produced significant accumulation of heavy metals in alfalfa roots, especially in AM-treated plants but no harmful effects on plant growth or nodule activity were apparent in this latter treatment (Fig. 1, Tables 3 and 4). Similarly, some studies have shown that sludge application could not reduce nodulation capacity of Sinorhizobium meliloti due to the fact that organic and mineral composition of sludge contained sufficient carbon, N, P and micronutrients to sustain bacterial growth (Rebah et al., 2007). In our study AM-amended soils had lower N and higher available P and K than ATAD-treated soils, and this could explain, at least in part, that nodulation and plant growth performed better in presence of AM sludge (Table 2). Our results agree with other authors, which showed that sludge addition did not affect nodulation, nodule glutamine synthetase activity or leghemoglobin content (Arau´jo et al., 2007). A potential metabolic constraint for nodule activity is the oxidative damage of nodule components (Dalton et al., 1986; Becana et al., 2000; Minchin et al., 2008) due to the presence of high amounts of heavy metals in root tissue (Table 3). This was evidenced in ATAD-treated nodules by enhanced nodule malondialdehyde (MDA) concentration (Table 5). The MDA, a major cytotoxic product of lipid peroxidation acts as an indicator for free radical production that occurs during different stresses, including heavy metals. In agreement with our data, other authors have shown increased tissue MDA in response to Cr (Sinha et al., 2005), Pb (Reddy et al., 2005), Cd (Carpena et al., 2003; Corticeiro et al., 2006) and sludge applications (Chandra et al., 2008). Regulation of antioxidant enzyme activities is an immediate and efficacious response to scavenge the ROS excess (Apel and Hirt, 2004). In this study we found that AM and ATAD-treated nodules exhibited enhanced antioxidant enzyme activities, highlighting the capacity of this tissue to scavenge ROS excess (Table 6) (Matamoros et al., 2003). However, this ability appears to be more successful in AM-treated nodules, which had the lowest H2O2 concentration (Table 5). Our data suggest that no harmful effects on rhizobia infection and nodule development in AM-treated plants could be related to improved thiol synthesis in nodules, which might have protected rhizobia from soil heavy metals (Table 7). Moreover, this accumulation of total thiols in nodules supports the hypothesis that a possible strategy of thiol sequestration of heavy metals is operating in alfalfa (Hall, 2002). As a consequence, no significant accumulation of MDA was found in AM-treated nodules in spite of increased root concentration of Cr, Cu and Ni (Tables 4 and 5). In the same way, Chandra et al. (2008) showed that sludge amended plants increased thiols levels. The relative abundance of ASC (high ASC/DHA) (Table 7) in the noduleinfected tissue of AM-treated plants in comparison with ATADtreated nodules strongly suggests that these redox metabolites cooperate in scavenging harmful concentrations of H2O2 in host cells by fueling the ASC–glutathione cycle. This pathway is critical for nodule functioning and it has been demonstrated that nodules are able to synthesize ASC (Matamoros et al., 2006).

5. Conclusions This study shows that the processing treatment for sewage sludge digestion results in diverse effects on plant growth and nodule establishment of alfalfa. Results showed that plant accumulation of heavy metals from high rates of sludge addition had imposed

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oxidative stress, which is characterized by induction of the activities of APX, GR and CAT and alterations in the redox state of ASC. ATAD sludge application produced a reduction in nodulation, increased nodule antioxidant enzyme activities and decreased ASC/DHA ratio. As a consequence, ATAD-treated nodules suffered some oxidative damages as evidenced by high MDA levels. By contrast, AM application enhanced plant growth and no deleterious effects on nodulation. Nodules developed in high rates of AM sludge had increased antioxidant enzyme activities and ASC/DHA ratio that could contribute to avoid oxidative damages. Moreover, only AM plants had improved capacity for thiol synthesis that might protect rhizobia from heavy metals present in roots. In our experimental conditions alfalfa performed better in AM than in ATAD sludge and this differential response appears to be mediated by plant ability to thiol synthesis and to maintenance of a more equilibrated antioxidant status.

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