Biochar impact on physiological and biochemical attributes of spinach Spinacia oleracea (L.) in nickel contaminated soil

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1 Glol J. Environ. Si. Mnge., (3): Glol , J. Environ. Summer Si. Mnge., 25 (3): , Summer 25 ISSN Biohr impt on physiologil nd iohemil ttriutes of spinh Spini olere (L.) in nikel ontminted soil U. Younis; 2* M. Athr; S. A. Mlik; M.H. Rz Shh; S. Mhmood Institute of Pure nd Applied Biology, Bhuddin Zkriy University, Multn, Pkistn 2 Cliforni Deprtment of Food nd Agriulture, 3288 Medowview Rod, Srmento, Cliforni, USA Reeived 6 Deemer 24; revised 2 Ferury 25; epted 24 Ferury 25; ville online June 25 ABSTRACT: Disstrous effet of nikel on spinh ws disussed y numer of uthors ut the effet of mendments like iohr with nikel on Spine olere L. is not still disussed y ny uthor of the world euse iohr ws used s soil mendments whih ply vitl role in reduing moiliztion nd uptke of nikel y spinh plnts. As nikel ontminted plnts re very hrmful for the onsumption y living orgnisms. Nikel n e gthered in gronomi soils y nthropogeni tions suh s Ni-Cd tteries. In this study, the growth, physiologil, photosyntheti nd iohemil responses of Spini olere grown in Ni-spiked soil (, 25, 5 nd soil) t three levels of otton-stiks-derived iohr (, 3 nd 5 %) were evluted. The results exposed signifint derese in growth, photosyntheti, physiologil, nd iohemil trits of S. olere when grown in Ni-polluted soil. However, this derese ws less pronouned in otton-stiks-derived iohr mended soil. A stedy rise in the MDA (.66 µg/g to 2.8 µg /g), sori id (.24 mg/g to.57 mg/g)nd sugr onentrtions (.73 mg/g to 2.6 mg/g)ws oserved with inresed onentrtion of Ni. The inresing perentges of otton-stiks-derived iohr from 3 % to 5 % deresed Ni onentrtions in root nd shoot of experimentl plnt. Higher prodution of hlorophyll, mino ids nd protein with otton-stiks-derived iohr mendment looked like llevition in Ni toxiity. Therefore, it is onluded tht, Ni toxiity nd vilility to the plnts n e redued y otton-stiks-derived iohr mendments. Keywords: Chrol, otton-stiks-derived iohr (CSB), Nikel toxiity, Pyrolysis, Spine olere INTRODUCTION Spinh (Spini olere) is the importnt lefy vegetle tht elongs to the fmily Amrnthee. Due to esy, pre-lened nd pre-pkged units the demnd of spinh mong the peoples of world is ontinuously inresing (Luier nd Plummer, 24). Hving rih soure et rotene, folte, vitmin C, lium, iron nd highest ORAC (Oxygen Rdil Asorne Cpity) spinh is highly onsumed ll over the world (Dioteu, 2). The ge relted mulr degenertion is lso redued due to the onsumption *Corresponding Author Emil: triq@df..gov Tel.: ; Fx: Note. This mnusript ws sumitted on Deemer 6, 24; pproved on Ferury 24, 25; pulished online on June, 25. Disussion period open until Otoer, 25; disussion n e performed online on the Wesite Show rtile setion for this rtile. This pper is prt of the Glol Journl of Environmentl Siene nd Mngement (GJESM). of spinh s it is rih soure of lutein i.e. rotenoid (Morelok nd Correll, 28). The growth of spinh is oserved est in sewge irrigted soil with rod leves. As result the quntity improved ut the qulity of spinh is destroyed due to hyper umultion of hevy metls in its shoot nd leves (Wgner nd Kupenjohnn, 24). The soure of hevy metls re nthropogeni tivities like mining nd industriliztion whih disrupted the nturl eosystem (Shh nd Nongkynrih, 27; Younis et l., 25). Among these metls whih re toxi for humns inlude P, As, Cd, Ni nd merury (Ahluwli nd Dinesh, 27). Nikel is the 24th most plentiful element of the erth rust (Cempel nd Nikel, 26). Sewge sludge in high mounts dds lrge quntities of Ni in soils tht re ner to the industries (Slt nd Krämer, 2). In soil the onentrtion of Ni umultion n

2 Glol J. Environ. Si. Mnge., (3): , Summer 25 U. Younis et l. e vried from 3- ppm (Sott-Fordsmnd, 997). Ni exists in soil in severl forms suh s rystlline minerls (inorgni), on tions exhnge surfes whih re inorgni, tions surfes tht re orgni, s free ion, wter solule nd s helted ompound (Sott-Fordsmnd, 997). Nikel is toxi to humn helth nd uses ner nd other diseses on onsumption of ontminted food. The edile prt of mny vegetles is the potentil sink of Ni where it umultes nd eomes toxi for ll the onsumers (Strug et l., 22). In plnts the toxiity effets used y Ni vry ording to the onentrtion of Ni in the soil solution (Mizuno, 968). The hlorosis nd nerosis re usully oserved in those plnts tht re suffering from high toxiity of Ni. Also it disturs the nturl mehnism of Iron (Fe) uptke nd mitoti tivity in the plnts whih dversely ffet their growth (Mdhv Ro nd Sresty, 2). Seed germintion is lso redued in mny plnts when they re ultivted on sites ontminted with Ni (Mdhv Ro nd Sresty, 2). In se of erels espeilly whet plnts, the shoot growth eomes retrded due to toxiity of Ni whih uses redution in yield (Gjewsket l., 26; Tripthi nd Gur, 26). Therefore, there is need to relim Ni in soil to redue its hzrdous impts on plnts nd nimls. For the relmtion of hevy metls through proess of immoiliztion in the soil mostly orgni nture mendments re suggested. In these orgni nture mendments iohr n prove very effetive nd n signifintly enhne the metls immoiliztion in the soils through whih metls iovilility n e redued (Verheijen et l., 2; Downie et l., 29). Reent studies show tht the pplition of iohr in the soil enhnes the soil s gronomi vlues s well s relims the orgni nd inorgni ontmintion through its high sorption pility (Asi et l., 29; Hossin et l., 2; Uhimiy et l., 22). The tive sites on the iohr due to presene of its funtionl groups lso mke it n effetive mendment for the relmtion of hevy metls in the soil. Hevy metls ind themselves with the tive sorption sites of iohr whih redue their moility in the soil (Mhid et l., 25). There is still gret unertinty mong the sientists regrding the influene of the iohr on produtivity of griulturl ommodities (Jh et l., 2). In the present study, moility nd iovilility of Ni y pplition of iohr nd its susequent effet on the growth of Spinh hve een studied. It is lso neessry to hek the mximum rte of iohr pplition in the soil whih would not mke it hrmful degrding gent. Spinh is seleted s n experimentl plnt due to its extensive onsumption s nutrient, pity to uptke nd store Ni ions. MATERIALS AND METHODS A pot experiment ws onduted t the Botnil Grden of Bhuddin Zkriy University Multn, using ompletely rndomized ftoril design involving spinh ( Spini olere) nd two tretment ftors: iohr (, 3, nd 5 % w/w) nd nikel dditions (, 25, 5 nd mg Ni/kg soil using NiSO 4 ). Eh tretment ws replited four times. The soure of otton-stiks-derived iohr (CSB) (Tle ) ws otton-stiks treted t 45 C in n espeilly designed pyrolysis retor. The plnts were grown in ly pots eh filled with mixture of 5 kg soil (Tle 2) nd the lulted mounts of respetive tretment ftors (CSB nd Ni). Seeds of Spini olere were purhsed from the lol mrket nd sowing ws done in ll the pots with their holes losed to prevent wshing nd lehing of Ni. At the seedling stge, plnts per pot were llowed to grow. The plnts were irrigted on regulr sis to mintin 5 % of wter holding pity. At mturity, the plnts were hrvested. Fresh nd dry iomsses were determined. The onentrtions of Ni in the plnt smples were determined using di-id digestion followed y using tomi sorption spetrophotometer equipped with grphite furne nd hydride/hydrea 34 tehnology, NovAA4, Anlyti Jen (Rshid, 986). Totl solule protein nd mino ids were determined following the proedures of Brdford (976). The onentrtion of sori id Tle : Physiohemil hrteristis of iohr derived from otton stiks ph EC (ds/m) Voltile mtter (%) Ash(%) Fixed ron (%) Nitrogen (%) Phosphorus (mg/kg) Potssium (mg/kg) Biohr

3 Glol J. Environ. Si. Mnge., (3): , Summer 25 Tle 2: Physiohemil hrteristis of experimentl soil Chrteristi ph s ( sturted pste) EC e TSS Wter Solule Crontes Wter Solule Birontes Wter Solule Chlorite Wter Solule Sulphte Wter Solule Clium + Mgnesium Wter Solule Sodium Orgni mtter Nikel Unit - ds/m % mg/kg Vlue ws determined using the formul of Keller nd Shwger, (977). Plnt mlondildihyde ontents were ssyed s thiorituri id (TBA) method nd solule sugr ws determined with the nthrone regent (Ckmk nd Horst, 99). Gs exhnge ttriutes (photosyntheti rte, trnspirtion rte (E) nd sustomtl CO2 onentrtion (Ci)) were determined using n open system LCA-4 ADC portle infrred gs nlyzer (Anlytil Development Compny, Hoddeson, Englnd). The photosyntheti pigments were determined following the method of Arnon (949). Sttistil nlysis The dt were nlyzed for sttistil differenes y performing nlysis of vrine (ANOVA) test using sttistil softwre pkge SPSS version 8.. The Tukey-HSD Test ws used to find signifint differenes etween vrious tretments. RESULTS AND DISCUSSION Growth Fig. is plotted y tking nikel onentrtions (, 25, 5, soil) on X-xis nd fresh nd dry iomss on Y-xis. It represented from the figure tht the fresh-iomss of S. olere ws greter t 3 % CSB pplition rte thn t 5 % CSB nd ontrol tretments. The inrese in Ni onentrtions indued negtive effet on fresh weight whih deresed from.69 g (ontrol) to g ( mg Ni /kg). However, the ddition of CSB with inresing levels (3% nd 5%) in the Ni-spiked soil signifintly enhned the fresh iomss nd redued the negtive impt of Ni (Fig. ). The dry iomss of experimentl plnts ws lso signifintly (P <.) deresed y the pplition of Ni levels from 25 mg Ni/kg to mg Ni/kg (Fig. ). However, the CSB pplition reveled signifint inrese of dry iomss t ll levels of Ni. The dry iomss inresed from 7.49 g (ontrol + mg Ni/kg) to.88 g with iohr pplition ( + mg Ni/kg). Spinh is soure of mny vitmins nd minerls like, A, B2, B6, C, E, K, folte, zin, selenium, opper, mgnesium, lium, iron nd potssium. It gives good supply of dietry fier nd omeg-3 ftty ids. Pollution of griulturl industries is min soure of ontmintion in spinh. So, the foremost purpose of this study ws to ssess the role of iohr on spinh in Ni-ontminted soil. For this, Spini olere ws seleted due to its hyper umulting ility of hevy metls. Similrly, iohr hs the ility to dsor Ni nd redued its toxiity. The Results of the study lerly indited n inresed in plnt weight with inrese of iohr pplition in ontrolled nd Ni ontminted soil (Fig. ). This inrese ws minly due to iohr improving the soil s overll physiohemil properties. Moreover, iohr improves the ion uptke nd wter-holding pity of soils, leding to inresed wter usge ility y the plnts (Uzomet l., 2). In our se, CSB redued Ni stress, resulting in improved plnt growth whih is redued in nikel stress. Mdhv Ro nd Sresty (2), oserved negtive impt of Ni on the fresh nd dry iomss of roots nd shoots of plnts due to Ni storge in the plnts ody prts (Pndey nd Shrm, 22). 247

4 Glol J. Environ. Biohr Si. nd Mnge., nikel impt (3): , on spinh Summer 25 Fig. : Fresh nd dry iomss of S. olere under different levels of nikel (, 25, 5 nd mg Ni/kg soil) nd otton stiks iohr (CSB, %, 3% nd 5 %). Physiologil nd photosyntheti The physiologil ttriutes of the plnts were signifintly influened y the pplition of CSB nd Ni (Fig. 2). The photosyntheti rte, trnspirtion rte nd su-stomtl CO 2 onentrtions inresed with inresing CSB perentges from 3% to 5%. By inresing the level of Ni (from 25 mg Ni/kg to mg Ni/kg), physiologil ttriutes deresed rpidly. However, this derese ws less pronouned in the medium ontining CSB. Similrly, Ni in the growth medium signifintly redued the hlorophyll, nd totl hlorophyll of S. olere (Tle 3) expeted for hlorophyll t 25 mg Ni/kg (.64 mg/g fresh weight) nd for hlorophyll (.49 mg/g fresh weight) t 5 mg Ni/kg. There ws signifint derese in the onentrtions of pigments with inresing Ni levels. Highest drop in totl hlorophyll ontents from.63 mg/g fresh weight (ontrol) to.25 mg/g fresh weight were oserved t mg Ni/kg. It ws oserved tht the CSB dditions used less derese of these pigments. The rotenoid ontents were lso ffeted in similr wy like hlorophyll. Similrly, the onentrtions of nthoynin nd lyopene gve tretment speifi response t vrious levels of Ni nd CSB. The photosyntheti prmeters serve s physiologil inditors for determining the effets of Ni toxiity (Monni et l., 2). The sustomtl CO 2 onentrtion, photosyntheti nd trnspirtion rtes deresed with inresing Ni levels ut with the use of BC, this derese ws hnged to n inrese (Fig. 2). Similr results regrding Ni-indued redutions in photosyntheti nd trnspirtion rtes in vrious plnts hve een reported (Seregin nd Ivnov, 2). These dereses my e due to derese in hlorophyll ontents, hlorophyll deprivtion, nd/or disintegrtion nd destrution of Ruiso with stomt. It ws oserved tht when plnts were ultivted in the Ni toxi soils, the Ni treted plnts showed the highest symptoms of hlorosis. These symptoms my hve ppered due to the ompetition etween the Ni with C nd Mg intke in the plnts. Less intke of Mg resulted in the defiieny of hlorophyll prodution in the plnts nd ultimtely photosynthesis is redued (Mols, 997). Seregin nd Ivnov (2), noted tht when plnts uptke lrge mount of Ni nd store it in the lmell regions, the PS II eome distured due to whih redution in the photosynthesis tkes ple. It lso hnges the struture of eletron rriers y hnging the plstoquinone Q A nd Fe to plstoquinone Q B (Krup nd Bszynski, 995). An inrese in hlorophyll ontents ws lso oserved with inresed iohr perentges, s oserved previously with Ni stress. The derese in photosyntheti pigments is due to the inhiition of the tivities of enzyme tht ply roles in the synthesis of these pigments, suh s δ- minolevulini id dehydrtse nd proto- 248

5 Glol J. Environ. Si. Mnge., (3): , Summer 25 2 ) % CSB 8 /se m l 6 o m 4 (µ 2 te r e ti 8 th n 6 sy to o 4 h P 2 Photosyntheti rte (µmol m/se) e ) % CSB /s 3.5 m o l 3 m (m 2.5 te r 2 n.5 irtio n sp r T.5 Trnspirtion rte (mmol m/se) 25 5 s n tio tr n e o n l) C o 2 O o l/m C m l t (µ m to -s u S Su-stomtl CO2 Conentrtions (µ mol/mol) % CSB 25 5 Nikel pplition ( - soil) Fig. 2: Photosyntheti rte, trnspirtion rte nd su-stomtl CO 2 onentrtions of S. olere under different levels of nikel (, 25, 5 nd mg Ni/kg soil) nd otton stiks iohr (CSB, %, 3% nd 5 %) on. The different letters on rs show signifint differenes (P <.) etween iohr pplition levels within nikel pplition. 249

6 Glol J. Environ. Si. Mnge., (3): , Summer 25 U. Younis et l. Tle 3: Photosyntheti ttriutes of S. olere under different levels of nikel (, 25, 5 nd soil) nd otton stiks iohr (CSB, %, 3% nd 5 %). The different letters within olumn show signifint differenes (P <.) etween iohr pplition levels within nikel pplition. Ni Tretments Biohr pplition (%) Chlorophyll Chlorophyll Totl Chlorophyll Crotenoids Lyopene mg/g Anthoynin (umol/ml) Control % CSB % CSB % CSB % CSB hlorophylli-deredutse. It ontrols the io mnufturing of pigments nd Clvin yle. Similrly, Somshekrih et l., (992) suggested tht the derese in the hl. nd hl. re resulted due to dmging of hloroplst y Ni. The inrese in rotenoid ontents in the present study is onsistent with the findings of Chnev et l., (29) who reported inresed rotenoid ontents in mize under hevy metl stress. Biohemil ttriutes The onentrtion of solule proteins in the leves ws signifintly ffeted y the pplition of Ni nd CSB (Fig. 3). The derese in protein onentrtion (5.37 mg/g to 6.46 mg/g) ws noted with inresing levels of Ni irrespetive of CSB pplition. Similr results were oserved for totl solule mino-ids. Lowest onentrtion (.33 mg/g) of totl solule mino ids ws noted t mg Ni/kg. The MDA ontents were lso signifintly (P <.) influened y Ni tretment long with CSB. By inresing Ni onentrtions the MDA ontents lso inresed from.67 µg/g (ontrol) to 2.7 µg/g ( mg Ni/kg). However, y the ddition of CSB, this inrese ws hnged into derese t vrious levels of Ni (2.7 µg/g to.4 µg/g t with 5% iohr). A similr pttern for the sugr nd sori id onentrtions ws oserved t different levels of Ni nd CSB. However, sugr onentrtion inresed more rpidly t mg Ni/kg s ompred to MDA nd sori id. Ni stress ws lso notied in the form of deresed solule proteins in the plnts. This derese in protein ontent might hve een due to the redution of de novo synthesis of enzymes (Fig. 3). High mount of Ni intke redues the prodution of proteins in the sunflower, mize s well s in soyen (Shrm nd Dhimn, 23). In the forms of enzymes mny ell funtions re performed y the proteins in the ody of plnts. The inrese in the onentrtions of metlinding omplexes (phytoheltins nd metllothioneins) with hevy metl pollution ws lso demonstrted y Inouhe, (25). The inrese in proteins with iohr pplition under Ni stress might hve resulted from the sme reson. Amino ids re ritilly importnt for metolism of plnts s these re onnetion etween ron nd nitrogen metolism. The synthesis of mino ids y plnts is n environmentlly ontrolled ftor; thus, plnts produe high quntities of mino ids under limited stress onditions (Pnt et l., 2). Similr results were oserved in our study. The mount of mino ids inresed t 25 soil. This inrese ws more visile t 5% iohr pplition with Ni stress. In mny plnts the oxidtion of polyunsturted ftty ids nmed s lipids is lso oserved when 25

7 Glol J. Environ. Si. Mnge., (3): , Summer 25 Asori id (mg/g) % CSB MDA (µg/g) % CSB Sugr (mg/g) % CSB % CSB Protein (mg/g) Amino id (mg/g) soil Fig. 3: Biohemil ttriutes (Asori id, MDA, sugr, mino ids nd protein) of S. olere under different levels of nikel (, 25, 5 nd mg Ni/kg soil) nd otton stiks iohr (CSB, %, 3% nd 5 %). The different letters on rs show signifint differenes (P <.) etween iohr pplition levels within nikel pplition. 25

8 Glol J. Environ. Biohr Si. nd Mnge., nikel impt (3): , on spinh Summer 25 they re ultivted in the Ni toxi soils. At the highest levels of nikel pplition, the onentrtions of MDA, sugrs, nd sori id were inresed (Fig. 3). The inreses in these onentrtions, espeilly MDA nd sori id, ply protetive roles ginst Ni stress. The inrese in MDA (polyunsturted ftty ids ) hs lso een oserved in different plnts with Ni tretment (Demirl nd Türkn, 25). Similrly the high onentrtion of Ni in the whet plnts lso inrese the synthesis of MDA s found y mny sientists (Gonnelli et l., 2). The inresed in sori id under Ni stress ws lso oserved y Mishr nd Choudhuri (999), who suggested the defensive role of sori id in Ni stress. However, these inreses were muh less in BC-treted soils, suggesting role for BC in reduing the toxiity of Ni y sorption it in soils. Nikel onentrtion in plnts The onentrtion of Ni in root nd shoot inresed signifintly (P<.) with inresing Ni pplition levels (Tle 4), though, this inrese ws more pronouned t rhizosphere level. Mximum vlue of Ni onentrtion in root (6.652 ppm) ws noted t mg Ni/kg followed y 5 mg Ni/kg (7.26 ppm), nd the Tle 4: Effet of vrious pplition levels of nikel (, 25, 5 nd mg Ni/kg soil) nd otton stiks iohr (CSB, %, 3% nd 5 %) on nikel onentrtion in S. olere. The different letters within olumn show signifint differenes (P <.) etween iohr pplition levels within nikel pplition. Ni Tretments Control 25 5 Biohr % ge % CSB % CSB % CSB % CSB Nikel onentrtions ( dry mss) Root Shoot minimum (.59 ppm) ws under. However the ddition of CSB in Ni pplied pots signifintly redued the Ni onentrtions in the roots nd shoots of respetive plnts. CONCLUSION The results of the present study lerly indited tht different levels of nikel hd negtive effets on growth, hlorophyll nd physiologil ttriutes of spinh plnts. Furthermore, the inresed in prodution of sori id, sugr nd MDA indite the dmge impose y nikel on plnts. Though, the otton stiks iohr hd reveled the pility to sor metl in the soil nd delimited trnsfer of nikel ions to the eril tissue. Similrly, iohr pplition lso enhned the growth nd photosyntheti ttriutes of spinh under nikel stress. Therefore, iohr n e hose s n mendment in onditions where irrigtion wter is impure with signifint quntity of nikel s well s for the opertion of unrestrined soils polluted with this metl. ACKNOWLEDGEMENTS This reserh onstitutes prt of Ph.D. thesis of the first uthor. The uthors knowledge the Higher Edution Commission Pkistn for finnil support 252

9 Glol J. Environ. Si. Mnge., (3): , Summer 25 for this reserh work nd Noumn Mirz of University of Melourne, Austrli for ritilly reviewing the mnusript. CONFLICT OF INTEREST The uthors delre tht there is no onflit of interests regrding the pulition of this mnusript. REFERENCES Ahluwli Srjeet, S.; Dinesh, G., (27). Miroil nd plnt derived iomss for removl of hevy metls from wstewter, Bioresoure Teh., (98): (5 pges). Arnon, D.I., (949). Copper enzymes in isolted hloroplsts. Polyphenoloxidse in Bet Vulgris, Plnt Physiol., (24): - 5 (5 pges). Asi, H.; Smson, B.; Stephn, H., (29). Biohr mendment tehniques for uplnd rie prodution in Northern Los. Soil physil properties, lef SPAD nd grin yield, Field Crop Res., (): 8-84 (4 pges). Brdford, M.M., (976). A rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye inding, Anl. Biohem., (72): (7 pges). Ckmk, I.; Horst, W.J., (99). 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10 Glol J. Environ. Si. Mnge., U. Younis (3): et l , Summer 25 Shrm, A.; Dhimn, A., (23). Nikel nd Cdmium Toxiity in Plnts, J. Phrmeut. Si. Innov., (2): 2-24 (5 pges). Somshekrih, B.V.; Pdmj, K.; Prsd, A.R.K., (992). Phytotoxiity of dmium ions on germinting seedlings of mung en ( Phseolus vulgris): Involvement of lipid peroxides in hlorophyll degrdtion, Physiol. Plnt, (85): (5 pges). Tripthi, B.N.; Gur, J.P., (26). Physiologil ehviour of Senedesmus sp.during exposure to elevted levels of Cu nd Zn nd fter withdrwl of metl stress, Protoplsm, (229): -9 (9 pges). Uhimiy, M.; Cntrell, K.B.; Hunt, P.G.; Novk, J.M.; Chng, S., (22). Retention of hevy metls in typi kndiudult mended with different mnure-sed iohrs, J. Env. Qul., (4): (2 pges). Younis, U.; Qyyum, M.F.; Shh, M.H.R.; Dnish, S.; Shhzd, A.N.; Mhmood, S.; Mlik, S.A., (25). Growth, survivl nd hevy metl (Cd nd Ni) uptke of spinh ( Spini olere) nd fenugreek ( Trigonell orniult) in iohr-mended sewge-irrigted ontminted soil, J. Plnt Nutr. Soil Si., DOI.2/jpln Uzom, K.C.; Inoue, M.; Andry, H.; Fujimki, H.; Zhoor, A.; Nishihr, E., (2). Effet of ow mnure iohr on mize produtivity under sndy soil ondition, Soil Use Mnge., (27): (8 pges). Verheijen, F.; Jeffery, S.; Bstos, A.C.; Vn der Velde, M.; Difs, I., (2). Biohr Applition to Soils: A Critil Sientifi Review of Effets of Soil Properties, Proesses nd Funtions. JRC Sientifi nd Tehnil Reports, EUR EN, Itly. Wgner, A.; Kupenjohnn, M., (24). Suitility of iohrs (pyro- nd hydrohrs) for metl immoiliztion on former sewge-field soils, Eurpen J. Soil Si., (65): ( pges). AUTHOR (S) BIOSKETCHES Younis, U., Ph.D.; Institute of Pure nd Applied Biology, Bhuddin Zkriy University, Multn, Pkistn. Emil: uzm.otny@hotmil.om Athr, M., Ph.D, DS., Professor; Cliforni Deprtment of Food nd Agriulture, 3288 Medowview Rod, Srmento, Cliforni, USA. Emil: thr.triq@df..gov Mlik, S.A., Ph.D., Professor; Institute of Pure nd Applied Biology, Bhuddin Zkriy University, Multn, Pkistn. Emil; seedotny@hotmil.om Rz Shh, M.H., MS. Student; Institute of Pure nd Applied Biology, Bhuddin Zkriy University, Multn, Pkistn. Emil: hrshh@hotmil.om Mhmood, S., Ph.D., Professor nd Diretor; Institute of Pure nd Applied Biology, Bhuddin Zkriy University, Multn, Pkistn. Emil: drseempk@gmil.om How to ite this rtile: (Hrvrd style) Younis, U.; Athr, M.; Mlik, S.A.; Rz Shh, M.H.; Mhmood, S., (25). Biohr impt on physiologil nd iohemil ttriutes of spinh Spini olere (L.) in nikel ontminted soil, Glol J. Environ. Si. Mnge., (3):