Nickel in a tropical soil treated with sewage sludge and cropped with maize in a long-term field study

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1 Soil Biology & Biochemistry 39 (2007) Nickel in tropicl soil treted with sewge sludge nd cropped with mize in long-term field study Wnderley Jose de Melo, Priscil de Stéfni Aguir, Gbriel Murı cio Peruc de Melo, Vle ri Peruc de Melo Lbortório de Biogeoquímic, Deprtmento de Tecnologi, Fculdde de Ciêncis Agráris e Veterináris, Universidde do Estdo de São Pulo. Vi de Acesso Prof. Pulo Donto Cstellne, Km 5. CEP Jboticbl, SP, Brzil Avilble online 26 December 2006 Abstrct Sewge sludge produced by the SABESP wstewter tretment plnt (Compnhi de Snemento B sico do Estdo de S o Pulo), locted in Brueri, SP, Brzil, my contin high contents of nickel (Ni), incresing the risk of ppliction to griculturl soils. An experiment ws crried out under field conditions in Jboticbl, SP, Brzil, with the objective of evluting the effects on soil properties nd on mize plnts of incresing rtes of sewge sludge rich in Ni tht hd been pplied for 6 consecutive yers. The experiment ws locted on Typic Hplorthox soil, using n experimentl design of rndomized blocks with four tretments (rtes of sewge sludge) nd five replictions. At the end of the experiment the ccumulted mounts of sewge sludge pplied were 0.0, 30.0, 60.0 nd 67.5 t h 1. Mize (Ze mys L.) ws the test plnt. Soil smples were collected 60 d fter sowing t depths of 0 20 cm for Ni studies nd from 0 to 10 cm nd from 10 to 20 cm for urese studies. Sewge sludge did not cuse toxicity or micronutrient deficiencies to mize plnts nd incresed grin production. Soil Ni ppered to be ssocited with the most stble frctions of the soil orgnic mtter nd ws protected ginst strong extrcting solutions such s concentrted nd hot HNO 3 nd HCl. Ni dded to the soil by sewge sludge incresed the metl concentrtion in the shoots, but not in the grin. The Mehlich 3 extrctor ws not efficient to evlute Ni phytovilbility to mize plnts. Soil urese ctivity ws incresed by sewge sludge only in the lyer where the residue ws pplied. r 2006 Elsevier Ltd. All rights reserved. Keywords: Sewge sludge; Nickel; Soil pollution; Mehlich 3; Urese; Phytovilbility 1. Introduction Sewge sludge is residue from domestic nd industril wstewter tretment. Incresing costs of commercil fertilizers nd lrge mounts of sewge sludge produced worldwide hve mde croplnd ppliction of this residue n ttrctive disposl option. Chemicl nd biologicl compositions of sewge sludges depend on the wstewter composition (Melo et l., 2002). Usully, it is rich in orgnic mtter (OM) nd plnt nutrients such s nitrogen (N), phosphorus (P) nd clcium (C) (Hue, 1988), nd cn improve soil physicl, chemicl nd biologicl properties, such s porosity, ggregte stbility, bulk density, soil Corresponding uthor. Tel.: x228; fx: E-mil ddress: wjmelo@fcv.unesp.br (W.J. de Melo). fertility, wter movement nd retention (Silveir et l., 2003). In the tropics, where soils re generlly poor in minerls with high ction exchnge cpcity (CEC) nd in OM, sewge sludge could be n importnt soil mendment (Melo et l., 2002). A problem to be solved is the hzrdous constituents, such s hevy metls, mong them nickel (Ni), nd persistent orgnic pollutnts (POPs) normlly present in sewge sludge. These my cuse soil nd wter pollution nd toxicity to crops, nimls, nd humns through the trophic chin (McBride, 1995). Ni concentrtions in sewge sludge vry widely, depending on the origin of the sludge nd on the processes of tretment in the sewge tretment plnt (Mttigod nd Pge, 1983); its uptke by plnts depends on the form in the soil nd on the plnt species. The legisltion regulting sewge sludge ppliction to soils generlly considers only the totl content of hevy metls in the residue. The legisltion /$ - see front mtter r 2006 Elsevier Ltd. All rights reserved. doi: /j.soilbio

2 1342 ARTICLE IN PRESS W.J. de Melo et l. / Soil Biology & Biochemistry 39 (2007) dopted by CETESB (Compnhi de Tecnologi e Snemento Ambientl) (1999) in S o Pulo Stte, which is the sme s tht of the Environmentl Protection Agency (EPA 503) legisltion for the United Stte of Americ, estblishes tht the upper concentrtion of Ni in sewge sludge for griculturl purposes is 420 mg kg 1 (dry weight bsis). Sewge sludge produced by SABESP (Compnhi de Snemento B sico do Estdo de S o Pulo), which trets the sludge produced in the Metropolitn Region of S o Pulo, sometimes exceeds tht limit. Considering tht Brzilin soils re quite different from US soils, the im of this work ws to estimte the effect of rtes of ppliction of sewge sludge produced by SABESP on the Ni forms in the soil nd on its potentil vilbility to mize (Ze mys L.) plnts cropped in sndy soil in field experiment crried out for 6 consecutive yers. 2. Mterils nd methods The experiment ws crried out under field conditions, using rndomized block design with four tretments (rtes of sewge sludge) nd five replictions, in Jboticbl, SP, Brzil ( S, W), from 1997 to A sndy soil (cly, 26%; silt, 7%; snd, 67% t depth of 0 to 20 cm) clssified s Typic Hplorthox, ws used for pplying sewge sludge nd mize ws cropped. In the first yer, the rtes of sewge sludge pplictions were 0.0 (control, with no sewge sludge nd no minerl fertiliztion), 2.5, 5.0 nd 10.0 t h 1 (dry weight bsis). Strting in the second yer, the control plots were fertilized with minerl fertilizers ccording to the soil chemicl nlysis nd the recommendtions of Rij et l. (1997). Strting in the fourth yer, the 2.5 rte ws replced by 20.0 t h 1 in order to increse the concentrtion of Ni in the soil. Therefore, the ccumulted mounts of sewge sludge over six yers were 0.0, 30.0, 60.0 nd 67.5 t h 1. The soil chemicl properties of the control plots before ech growing seson re shown in Tble 1. Sewge sludge ws obtined from the sewge tretment plnt operted by SABESP, locted in Brueri, SP, nd its chemicl composition in the 6-yr experiment is presented in Tble 2. Totl N in sewge sludge ws determined by the microkjeldhl method, potssium (K) by flme photometry, P by the vndo-molybdte spectrophotometric method, nd the hevy metls, including Ni, by tomic bsorption spectrometry (AAS) in the extrct obtined by digestion with HNO 3 +H 2 O 2 +HCl (USEPA, 1986). Before strting the experiment (September, 1997), the re ws plowed, hrrowed, nd limed with 2.5 t h 1 of dolomitic limestone to rise the bse sturtion (BS) to 70%. Thirty dys fter liming, the plots were mrked out (54 m 2 ech nd 28.8 m 2 of useful re for soil nd plnt smpling), including six 10-m long rows, spced 0.90 m prt; the two centrl rows were reserved for evlution of grin production, nd the others for plnt nd soil smpling. Sewge sludge (77.6% moisture) ws spred on the soil surfce nnully nd incorported into the top10 cm lyer with rotry hoe (first yer), or light hrrowing (lst five yers). Plots treted with sewge sludge received supplementry P nd K s minerl fertilizers, when necessry (Tble 3), in order to supply them with the sme mounts of NPK s the control. The minerl fertilizer ws pplied in the plow furrow. The minerl fertiliztion pplied to the control plots in the 6 yers of experimenttion is shown in Tble 3. N fertiliztion of the control plots ws divided nd the side dressing ws pplied 45 d fter sowing. The test plnt in ll 6 yr ws mize (Ze mys L.), grown t density of 5 7 plnts m 1 (bout 55,000 plnts h 1 ). Plnt smples (5 plnts per plot) were tken 100 d fter sowing. The plnts were cut just bove the soil surfce, wshed with diluted neutrl detergent, wter, distilled wter, deionized wter, dried t 70 1C in forced ir oven, ground nd, fter nitric-perchloric digestion, the extrct ws nlyzed for totl Ni by AAS (Btgli et l., 1983). For evlution of crop production, plnts were hrvested 120 d fter sowing from the two centrl rows of ech plot, dried t 65 1C in forced ir oven, nd weighed. The dt for grin production were corrected to 13% moisture. Ni determintion in the grin ws processed by the sme method described for the shoots. Soil smples (0 20 cm lyer) for Ni nlysis were collected 60 d fter sowing in the sixth yer of experimenttion. In the Tble 1 Chemicl properties in the 0 20 cm lyer of the control plots of Typic Hplorthox soil before sewge sludge ppliction in ech of the 6 yr of experimenttion Yer ph CCl 2 O.M. (g dm 3 ) P resin (mg dm 3 ) K C Mg H+Al SB b CEC BS c (%) 1997/ / / / / / Vlues on n ir dried bsis. b SB, sum of bses. c BS, bse sturtion.

3 W.J. de Melo et l. / Soil Biology & Biochemistry 39 (2007) Tble 2 Chemicl composition of sewge sludge in ech of the six yers of experimenttion Element Unit 1997/ / / / / /03 Nitrogen g kg Phosphorus g kg Potssium g kg Mngnese mg kg Zinc mg kg Copper mg kg Nickel mg kg Led mg kg Cdmium mg kg Chromium mg kg Vlues on n ir dried bsis. Tble 3 Chemicl fertiliztion pplied to Typic Hplorthox soil cropped with mize during the six yers of experimenttion Growing seson Rte Nutrient (kg h 1 ) 1997/ / / / / /03 Sowing stge. b Side-dressing stge. N P 2 O 5 K 2 O S D b S S D (20) (20) (20) re reserved for soil smpling of ech plot, 12 single smples were collected 10 cm from the sowing furrow, t the side of mize plnt, nd composited. Soil smples were ir dried nd sieved (2 mm) before chemicl nlysis. For urese ctivity evlution, soil smples were collected from the lyers 0 10 nd cm. Totl Ni ws determined by AAS on the extrct obtined by the two methods. In the USEPA (1986) method, since nmed the USEPA method, the extrct ws obtined s follows: the soil smple (2.00 g) ws plced in 100 ml beker nd 10 ml of 1+1 (v/v) HNO 3 were dded, followed by heting on hot plte t C for 15 min. After heting, concentrted HNO 3 (6 ml) ws dded nd heted for further 30 min. This opertion ws repeted until no more brown fumes were formed nd the volume of the smple reched bout 5 ml. Deionized wter (2 ml) nd 3 ml of 30% (w/w) H 2 O 2 were dded nd the smple ws heted with repeted dditions of 1 ml of H 2 O 2 (mximum of 10 ml) until the rection stopped nd the volume of the extrct reched bout 5 ml. Then concentrted nitric cid (10 ml of 35%, w/w) ws dded nd the extrct heted t C for 15 min. The extrct ws trnsferred to 50 ml volumetric flsk nd mde up to volume with deionized wter. In the method described in Jckson (1958), since nmed the Jckson method, the soil extrct ws obtined s follows: Soil (100 mg) ws plced in 50 ml Teflon crucible. The smple ws wetted with few drops of deionized wter. Then, 0.5 ml of concentrted HClO 4 nd 5 ml of 48% HF were dded. The crucible, with the lid covering nine-tenths of the top, ws plced on snd bth t temperture of C, nd the cids were evported to dryness. The crucible ws then removed from the snd bth, cooled, 6 M HCl (5 ml) ws dded nd the suspension ws diluted with 15 ml of deionized wter. The crucible ws then put on hot plte nd the suspension boiled gently for 5 min. When the soil ws totlly dissolved, the solution ws cooled, trnsferred to 100 ml volumetric flsk nd mde up to volume with deionized wter. Soil orgnic mtter (SOM) ws extrcted nd frctionted by modified method of Dbin (1971), which consisted of extrcting the humic substnces (HS) from the soil smples with 0.1 M NOH, followed by cidifying the lkline extrct to ph 1 with concentrted H 2 SO 4. The soluble frction ws the fulvic cids (FAs) nd the precipitted frction ws the humic cids (HAs). Ni ws determined by AAS in the extrcts obtined from the different frctions by the USEPA method. Ni potentilly vilble to mize plnts ws estimted by the Mehlich 3 extrction method (Mehlich, 1984). Soil

4 1344 ARTICLE IN PRESS W.J. de Melo et l. / Soil Biology & Biochemistry 39 (2007) (5.00 g) ws plced in 125 ml Erlenmeyer flsk nd 25 ml of Mehlich 3 extrctor were dded. The flsk ws shken for 5 min nd the suspension filtered through Whtmn number 1 filter pper. Ni ws determined in the extrct by AAS. A control ws crried out in the bsence of soil. Soil urese ctivity ws evluted by the method of My nd Dougls (1976) with the modifictions of Longo nd Melo (2005). Soil (30.00 g) ws plced in 125 ml Erlenmeyer flsk nd held t 30 1C for 10 min with 0.5 ml of toluol nd 12 ml of deionized wter. Then ure solution (3 ml of 3.5 g l 1 ) ws dded nd new incubtion of 60 min t 30 1C nd gitted. After incubtion, 2 M KCl (15 ml) contining phenylmercury cette (5 Mm) ws dded nd the filtrte (10 ml) ws used for NH 4 -N determintion by the stem distilltion method (Bremner nd Keeney, 1965). A control ws crried out in the sme wy, but ure solution ws dded fter the 60-min incubtion. The dt were nlyzed for vrince nd when the F-test ws significnt t Po0.01, the Tukey s test (Po0.05) ws used for comprison of mens. The correltion coefficients (R) between Ni extrcted from the soil nd the mounts tken up by mize plnts (grin+shoots) were clculted (Pimentel Gomes, 1966). 3. Results 3.1. Soil properties nd sewge sludge composition The Typic Hplorthox soil ws selected for this study becuse it occurs in lrge res in the Stte of S o Pulo. The chemicl properties of smples obtined from the control plots for the 6 yr of experimenttion re presented in Tble 1. These dt were used to clculte the minerl fertiliztion to be pplied to those plots. The sewge sludge generted in the sewge tretment plnt operted by SABESP in Brueri, SP, is rich in OM, plnt nutrients (except K) nd lso in hevy metls such s Ni, since the plnt trets the sludge collected from highly industrilized region (Tble 2). The content of Ni rnged from 231 (2002/2003) to 595 mg kg 1 (1998/1999), vlues sometimes higher or very close to the limit estblished by CETESB (1999), where sewge sludge is pplied to griculturl res Forms of Ni in soil Totl soil Ni, estimted ccording to the USEPA method (Tble 4), rnged from 6.40 (control) to mg kg 1 (in the 60.0 t h 1 ccumulted sewge sludge tretment) while the totl soil Ni evluted ccording to the Jckson (1958) method rnged from (control) to mg kg 1 (in the 60.0 t h 1 ccumulted sewge sludge tretment). When totl Ni ws determined by the Jckson method, ll the dt obtined from tretments tht received sewge sludge differed from the control, but no difference ws detected mong the tretments tht received the sludge. Soil Ni extrcted by the Mehlich 3 extrctor method rnged from 0.35 (control) to 3.65 mg kg 1 (60.0 t h 1 sewge sludge plots). This is low frction of the Ni evluted by both the methods used for estimting totl Ni (23.4% nd 9.0% relted to the methods of USEPA nd Jckson, respectively). The mjor prt of the soil Ni ssocited with OM ws found in the humin frction (Tble 5), where concentrtion incresed from 4.10 (control) to mg kg 1 (60.0 t h 1 sewge sludge plots). This vlue is 3.4 times the content in the control. Ni in the FA frction incresed from 2.07 (control) to 2.89 mg kg 1 (67.5 t h 1 sewge sludge plots), nd Ni in the HA frction decresed from 0.23 (control) to 0.18 mg kg 1 (60 t h 1 sewge sludge plots). For the most prt, the Ni dded to the soil by sewge sludge ppered s humin, the most stble frction of the SOM. The distribution of Ni in the SOM is humin4fa4ha. This fct is importnt to consider becuse of the solubility of these soil constituents. It is lso importnt to observe tht the concentrtions of Ni extrcted by the Mehlich 3 extrctor nd the Ni present s FA were very close Plnt growth nd Ni uptke Mize plnts showed norml growth in the field. They did not present ny symptoms of Ni toxicity or micronutrient deficiency induced by n excess of Ni in the soil. Tble 4 Forms of Ni in Typic Hplorthox soil treted with sewge sludge nd cropped with mize for 6 consecutive yers Sewge sludge Totl Ni Mehlich 3 (mg kg 1 soil ) USEPA Jckson (mg kg 1 soil ) (mg kg 1 soil ) d b 17.28b 0.35c c b b Vlues on n ir dried bsis. b Mens followed by the sme letters in the sme column re not different by the Tukey s test t Po0.05. Tble 5 Ni in the soil orgnic mtter frctions of Typic Hplorthox soil treted with sewge sludge nd cropped with mize for 6 consecutive yers Sewge sludge Humin Ni (mg kg 1 soil ) Fulvic cid Ni (mg kg 1 soil ) d b 2.07c 0.23b c 2.32bc 0.05b bc 0.18b b Humic cid Ni (mg kg 1 soil ) Vlues on n ir-dried bsis. b Mens followed by the sme letters in the sme column re not different by the Tukey s test t Po0.05.

5 W.J. de Melo et l. / Soil Biology & Biochemistry 39 (2007) Dry mtter production by the eril prt of the mize plnts smpled 100 d fter sowing ws not ffected by the tretments (Tble 6), but the grin production incresed from the control (5.57 t h 1 t 13% moisture) to the highest rte of sewge sludge (8.60 t h 1 t 13% moisture), n increse of 3.03 t h 1. Sewge sludge incresed the Ni concentrtion in mize shoots (Tble 6) from 6.28 (control) to mg kg 1 (67.5 t h 1 sewge sludge), but it did not ffect Ni concentrtion in the mize grin Urese ctivity Soil urese ctivity rnged from 6.52 (control) to mg NH 4 -N kg 1 h 1 (67.5 t h 1 sewge sludge) in the smples obtined from 0 to10 cm lyer. For the smples obtined from 10 to 20 cm lyer, urese ctivity initilly decresed from (control) to 8.91 (60.0 t h 1 sewge sludge) nd then incresed to NH 4 -N kg 1 h 1 (67.5 t h 1 sewge sludge) s cn be seen in Fig. 1. The enzyme ctivity ws ffected differently by the tretments depending on the smpling depth. For smples Tble 6 Dry mtter, grin production nd grin Ni content in mize plnts cropped in Typic Hplorthox soil treted with incresing rtes of sewge sludge for 6 consecutive yers Sewge sludge Shoots Dry mtter Ni (mg kg 1 ) Grin b Mss Ni (mg kg 1 ) c 6.28b 5.57b ,87b 6.19b ,11b 7.20b , Vlues on n oven dried bsis. b Dt djusted to 13% moisture. c Mens followed by the sme letters in the sme column re not different by the Tukey s test t Po0.05. Ni (mg plnt -1 ) y = x R 2 = Ni mg kg -1 (Mehlich 3) Fig. 1. Determintion coefficients between Ni extrcted by the Mehlich 3 extrctor nd the metl bsorbed by mize plnts cropped in Typic Hplorthox soil treted with incresing rtes of sewge sludge for 6 consecutive yers. tken from the upper lyer, the lyer where sewge sludge ws incorported, urese ctivity incresed with the rte of sludge ppliction. On the other hnd, for the smples obtined from 10 to 20 cm lyer, the enzyme ctivity ws not ffected by the tretments. 4. Discussion 4.1. Soil properties nd sewge sludge composition The chemicl properties of the soil used in this experiment (Tble 1) clssify it s soil with medium fertility ccording to the concentrtions estblished in Rij et l. (1997). The Ni content in sewge sludge (Tble 2), in the second yer of experimenttion (1998/1999), it ws higher thn the 420 mg kg 1 (dry weight bsis) estblished by CETESB (1999) in its legisltion regulting sludge ppliction to griculturl soils. However, in most of the yers, the concentrtion of Ni in the sewge sludges ws very close to tht limit, nd so Ni is hevy metl tht cn limit the ppliction to griculturl soils. of the sewge sludges generted by the SABESP source. As the limiting vlue of 420 mg kg 1 ws not estblished for tropicl soils, it is very importnt to know the Ni behvior in soil when sewge sludge is dded, the forms in which it occurs, the vilbility to plnts, nd the trnsloction to the grin in order to prepre guidelines for pplictions tht will hve low risk for the environment Forms of Ni in soil Sewge sludge ppliction to the soil significntly incresed totl Ni content determined by the USEPA (1986) method up to the rte of 60.0 t h 1 nd then decresed the rte of 67.5 t h 1 (Tble 4). It is importnt to consider tht the ccumulted rte of 60.0 t h 1 ws obtined fter 6 yr of pplictions of 10 t h 1, while the ccumulted rte 67.5 t h 1 ws obtined in shorter time, since 60.0 t h 1 ws pplied in the lst 3 yers. This finding shows tht the interction of the components of sewge sludge with soil frctions is time dependent nd tht the USEPA method is not ble to mesure some forms of Ni in soil. Other uthors, estimting totl Ni by the USEPA method hve lso found n increse in totl soil Ni by incresing rtes of sewge sludge (F.C. Oliveir, unpublished Ph.D. Thesis, ESALQ, S o Pulo University, Pircicb, SP, Brzil, 2000; M. Mrchiori Jr, unpublished Ph.D. Thesis, FCAV, University of the Stte of S o Pulo, Jboticbl, SP, Brzil, 2002). When totl Ni ws mesured by the method of Jckson (1958), ll the rtes of sewge sludge showed n increse in the Ni concentrtion s compred to the control, but the different rtes of sewge sludge did not show different totls of soil Ni concentrtions. The USEPA method extrcted bout 47% of the Ni extrcted by the Jckson method. Thus the Ni extrcted by the USEPA method relly is not the totl Ni.

6 1346 ARTICLE IN PRESS W.J. de Melo et l. / Soil Biology & Biochemistry 39 (2007) One possible explntion for this finding is tht hevy metls in soils my be occluded in iron hydroxides nd mngnese oxides, in which tropicl soils re rich (Mlvolt, 1994). Wheres the USEPA method does not dissolve the entire soil smple, the Jckson method does. Mehlich (1984) proposed vrition of the Mehlich 1 extrctor, which is mixture of two diluted cids (H 2 SO 4 +HCl) to estimte the vilbility of hevy metls present in soils. This extrctor lso includes complexing gents nd is known s the Mehlich 3 extrctor. In our study, the ppliction of sewge sludge to Typic Hplorthox soil incresed extrctble Ni up to n ccumulted rte of 60.0 t h 1 nd then decresed t the highest ccumulted rte. This behvior is similr to tht obtined for totl Ni evluted by USEPA (1986), confirming tht t tht rte, ccumulted in shorter time, the content of esily extrctble Ni is lower. Other uthors, like Mrtins et l. (2003), lso hve observed tht incresing sewge sludge rtes induced liner increse in the Ni concentrtion of soil extrcts obtined with the Mehlich 3 extrctor. Ni distribution in the frctions of the SOM ws in the following order: humin4fa4ha. Considering tht the FA frction is soluble both in cid nd in lkline medi, it my be n importnt frction for the vilbility of Ni to plnts or for incresing the movement of the metl down in the soil profile. On the other hnd, Ni ssocited with humin, frction tht is not extrcted by n lkline solution, my be gretly unvilble to plnts nd wy to mintin Ni in the upper soil lyers. The findings tht soil Ni is not totlly extrcted by the USEPA method, nd tht soil Ni is concentrted in the humin frction of the SOM, cn explin why most uthors hve found tht Ni ccumultes in the top lyers of the soil. For exmple, Sidl et l. (2003) found tht more thn 93% of ll hevy metls pplied over 2-yr period were ccumulted in the surfce soil. Erlier, Anderson nd Nilson (1972) observed tht prcticlly ll the Ni remined in the 0 to 20 cm lyer of the soil, fter 12 yr of dding 84 t h 1 of sludge. The ccumultion of Ni in the top lyer of the soil cn be ttributed to the high ffinity of the metl to OM (McGrth nd Lne, 1989). Hevy metls like Ni my be dsorbed strongly to the soil orgnic nd minerl colloids like cly, iron hydroxides nd mngnese oxides nd to SOM (Chney nd Giordno, 1977) Plnt growth nd Ni uptke Metls nd other toxic products did not ffect mize plnt growth fter n ppliction of 67.5 t h 1 of sewge sludge in period of 6 consecutive yers. The plnts did not present ny symptoms of Ni toxicity or of deficiency in other nutrients cused by the presence of n excess of Ni in the soil. Dry mtter content of the shoots ws not ffected (Tble 6), but the grin production incresed in the presence of sewge sludge. This increse in grin production my be explined by the improvement in soil properties due to the OM nd plnt nutrients present in sewge sludge (Melo et l., 2002). From nlysis of mize uptke of Ni, nd the distribution of the metl in the plnt prts, we cn see tht sewge sludge incresed the metl content in the shoots (Tble 6). This ws not observed in the cse of the grin, which shows tht the trnsloction of Ni from leves nd stem to grin ws not significnt. In n experiment with Cedrel fissilis Vell, submitted to incresing rtes of Ni in the nutrient solution, Piv et l. (2002) found tht Ni concentrtions incresed in roots, stems nd leves nd tht the metl concentrtion ws higher in shoots thn in the grin. Miller et l. (1995), in n experiment with brley, lso observed tht the contents of Ni were higher in the strw thn in the grin. Anjos nd Mttizzo (2000) observed similr results for different prts of mize plnts cropped in Oxisols treted with sewge sludge. The increse in Ni concentrtion in stem+leves is not importnt for humn nutrition, but it is importnt for niml nutrition, if the mteril is used for silge or hy production. The Mehlich 3 extrctor ws not sufficiently good for estimting the vilbility of Ni to mize plnts (R 2 ¼ 0.56), s cn be seen in Fig. 2. Other uthors, such s Hq et l. (1980), Abreu et l. (1995), nd Anjos nd Mttizzo (2000) hve lso found tht the Mehlich 3 extrctor is not efficient for mesuring soil Ni vilbility to plnts. According to Pires nd Mttizzo (2003), the efficiency of this extrctor depends on the metl to be mesured, the type of soil, nd the plnt species Urese ctivity Longo nd Melo (2005b), studying the effect of smpling time, smpling depth nd the soil coverge, obtined urese ctivities vrying from 2 to 15 mg NH 4 -N dm 3 of soil h 1. They observed tht the enzyme ctivity ws ffected by the time of smpling, the highest ctivity being found in hot nd riny months, by the type of soil coverge, nd by the soil depth. mg NH 4 + kg -1 h B 0-10 cm cm B AB Accumulted Sewge Sludge (t h -1 ) Fig. 2. Urese ctivity in Typic Hplorthox soil treted with sewge sludge nd cropped with mize for 6 consecutive yers. Mens followed by the sme cpitl letter (0 10 cm) nd lower cse letter (10 20 cm) re not different by the Tukey s test t Po0.05. A

7 W.J. de Melo et l. / Soil Biology & Biochemistry 39 (2007) Urese is n enzyme tht depends on Ni for its ctivity. Thus it is possible to suppose tht the ddition of sewge sludge contminted with the metl might cuse n increse in the enzyme ctivity, if other pollutnts would not be ble to inctivte the enzyme. In this experiment, it ws observed tht sewge sludge incresed urese ctivity, when the smples were obtined from 0 to 10 cm lyer (Fig. 1), where the residue ws incorported. This increse in urese ctivity cused by sewge sludge ppliction my be explined by the content of OM, the types of plnts, nd the microorgnisms in sewge sludge, nd lso by the ure, the substrte of the enzyme, nd by some urese lredy present in sewge sludge. Nickel my lso hve contributed to tht increse, since the enzyme is metl-dependent. The fct tht the enzyme ctivity ws not ffected by the tretments in the smples obtined from 10 to 20 cm lyer suggests tht OM nd other fctors tht ffect urese ctivity, such s Ni, did not rech tht depth Conclusions Sewge sludge did not cuse toxicity, did not induce micronutrient deficiencies to mize plnts, nd did not ffect shoot dry mtter production, but it did increse grin production. Ni dded to the soil by sewge sludge ppered to be ssocited with the most stble frctions of the SOM nd protected ginst strong extrcting gents such s concentrted nd hot HNO 3 nd HCl, justifying its concentrtion in the upper soil lyers. Soil Ni fter 6 yr of experimenttion ws vilble to mize plnts, with incresed concentrtion in the shoots, but not in the grin. The Mehlich 3 extrctor ws not efficient to evlute Ni phytovilbility to mize plnts. Soil urese ctivity ws incresed by sewge sludge only in the lyer where the sludge ws pplied. 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