The Influence of Nitrogen on the Biological Properties of Soil Contaminated with Zinc

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1 Bull Environ Contm Toxiol (7) 98:46 4 DOI.7/s The Influene of Nitrogen on the Biologil Properties of Soil Contminte with Zin Rfł Strhel Jwig Wyszkowsk Młgorzt Bćmg Reeive: 8 July 6 / Aepte: 6 Novemer 6 / Pulishe online: Novemer 6 The Author(s) 6. This rtile is pulishe with open ess t Springerlink.om Astrt This stuy nlyze the reltionship etween nitrogen fertiliztion n the iologil properties of soil ontminte with zin. The influene of vrious onentrtions of zin n nitrogen on the miroiologil n iohemil tivity of soil ws investigte. In lortory experiment, lomy sn with ph KCl 5.6 ws ontminte with zin (ZnCl ) n fertilize with ure s soure of nitrogen. The tivity of i phosphtse, lkline phosphtse, urese n β-gluosise, n miroil ounts were etermine in soil smples fter n weeks of inution. Zin generlly stimulte hyrolse tivity, ut the highest zin ose (5 mg kg ) le to the inhiition of hyrolses. Nitrogen ws not highly effetive in neutrlizing zin s negtive effet on enzyme tivity, ut it stimulte the growth of soil-welling miroorgnisms. The hnges in soil iity oserve fter the ition of ure moifie the struture of miroil ommunities. Keywors Zin Soil Hyrolses Miroorgnisms Nitrogen Biostimultion Soil is ruil element of the iosphere, n it plys very importnt role in griulturl proution. Toxi ompouns re neutrlize in the soil environment where * Jwig Wyszkowsk jwig.wyszkowsk@uwm.eu.pl Rfł Strhel rfl.strhel@uwm.eu.pl Młgorzt Bćmg m.mg@uwm.eu.pl Deprtment of Miroiology, University of Wrmi n Mzury, Pl Łózki, 77 Olsztyn, Poln vrious iologil proesses enhne the iogeohemil yling of elements (Vrščj et l. 8). Most of those proesses involve miroorgnisms tht immoilize iogeni elements suh s ron, nitrogen n phosphorus. Soil miroiologil onsorti ply key role in the yling of sustnes in nture. Exessive umultion of toxi sustnes, suh s hevy metls, isrupts miroil evelopment (Arino et l. 4; Knopf n König ; Strhel et l. 6). Sine the eginning of the inustril revolution, pollution with hevy metls hs elerte rmtilly, posing sustntil risk for eosystem helth, in prtiulr in griulturl proution (Jing et l. 7). Zin is less toxi thn other hevy metls (C, Hg, P, As), ut exessive umultion of this element in the soil eosystem n exert negtive influene on soil miroorgnisms whih re the min eterminnts of soil fertility (MLughlin et l. 999). Zin ontmintion ereses the quntity n qulity of rops grown in res pollute with this metl (Chiuike n Oior 4; Yng et l. 6; Wyszkowsk et l. 6). Nitrogen fertiliztion signifintly improves the vilility of nitrogen in soil, n it stimultes the growth of soil-welling miroorgnisms. The miroiologil iversity of soil is onitione y nutrient vilility, n it is signifintly influene y fertiliztion (Wng et l. 5). The iohemil tivity of soil is relile initor of fertility, n it n hnge uner exposure to toxi sustnes (Chperon n Suvé 7; Kızılky et l. 4). Hyrolse tivity, roust mesure of soil helth, is influene y the ounts, omposition n enzymti potentil of soil-welling miroorgnisms, exposure to protetive tretments or sustnes tht inhiit enzyme tivity. This stuy evlute the effets of nitrogen fertiliztion in zin-ontminte soil on the tivity of selete hyrolses: i phosphtse, lkline phosphtse, urese n β-gluosise, n on the ounts of selete groups of

2 Bull Environ Contm Toxiol (7) 98:46 4 soil-welling miroorgnisms. Those enzymes re relile initors of soil qulity euse they prtiipte iretly in the yling of elements (ron, nitrogen n phosphorus) n respon ifferently to stressors, inluing zin. In the present stuy, ure ws pplie s the only soure of nitrogen to evlute its iostimultory effet on soil enzymes n miroorgnisms. Mterils n Methos Soil smples for the experiment were otine from the Agriulturl Experiment Sttion in Tomszkowo (north-estern Poln, 5.76 N,.467 E) operte y the University of Wrmi n Mzury in Olsztyn. Smples ollete from the rle lyer (epth of m) h the grnulometri omposition of lomy sn n were lssifie s Eutri Cmisols oring to the Worl Referene Bse of Soil Resoures (4). Before the experiment, soil ws thoroughly mixe n selete physiohemil properties of soil were etermine. Soil smples h the following prmeters: ph (in Mol KCl m ) 5.6 ±., C org. ±. g kg soil DM, N totl.58 ±,7 g kg soil DM, ville tions (mg kg soil DM): P 96. ±.48, K 79.8 ± 5.8, Mg 5.7 ± 8.; totl exhngele tions (mg kg soil DM): K + 7.7, C , N +.4, Mg ; hyrolyti iity (HAC) 8.66 ±.4 mmol (+) kg soil DM; totl exhngele ses (TEB) 4. ±. mmol (+) kg soil DM; tion exhnge pity (CEC) ±.64 mm(+) kg soil DM; se sturtion (BS) 68.9% ±.55; Zn totl.68 ±.7 mg kg DM soil; Zn ioville 9. ±.9 mg kg soil DM. The experiment ws rrie out in 5 m glss ekers. A ifferent set of ekers ws use for every nlytil te, whih proue totl of 8 ekers (six zin oses three nitrogen oses two nlytil tes three replitions). Soil smples of g (prtile size. mm) were omine with ZnCl in oses of, 5, 5, 75, n 5 mg Zn + kg soil DM. Nitrogen s influene on the iologil properties of zin-ontminte soil ws etermine y fertilizing soil smples with ure t the rte of, 5 n 5 mg N kg soil DM. Soil smples were inute for n weeks t temperture of 5 C n t 5% pillry wter pity. After n weeks of inution, the tivity of i phosphtse, lkline phosphtse, urese n β-gluosise ws etermine olorimetrilly y the metho propose y Alef n Nnnipieri (998). The nlyses were rrie out in triplite. The following sustrtes were use in enzyme tivity tests: 4-nitrophenyl phosphte isoium (for i phosphtse n lkline 47 phosphtse, λ = 4 nm), % ure solution (for urese, λ = 4 nm), n p-nitrophenyl-β-d-gluopyrnosie (for β-gluosise, λ = 4). The tivity of i phosphtse, lkline phosphtse n β-gluosise ws expresse in mmol 4-nitrophenol (PNP) kg soil DM h, n the tivity of urese in mmol N-NH 4 kg soil DM h. The nlyses were performe with the use of the Ceil Aqurius 75 spetrophotometer with wvelength rnge of 9 nm n ury of.5 nm. The ury of mesurements ws s follows: tivity of i phosphtse n lkline phosphtse.4 µm PNP, tivity of β-gluosise. µm PNP, tivity of urese. mm N-NH 4. Miroil ounts were etermine y the strek plte metho in three replitions. Orgnotrophi teri were ulture on soil extrt with the ition of Bunt n Rovir s meium (955), n oligotrophi n opiotrophi teri were grown on sustrte ontining peptone n met extrt (Ont n Httori 98). Atinomyetes were ulture on Küster n Willims meium with the ition of ntiiotis nysttin n tiione (Prkinson et l. 97). Fungl ounts were etermine on gluose peptone gr with the ition of rose engl n ureomyin oring to Mrtin s metho (95). Selete physiohemil prmeters of soil were nlyze fter weeks of inution (in triplite). Soil ph ws etermine potentiometrilly in M KCl queous solution; hyrolyti iity (HAC), totl exhngele ses (TEB), tion exhnge pity (CEC) n se sturtion (BS) were etermine oring to the methos esrie y Crter (99). Soil smples (fter weeks inution) were ssye for ioville zin n totl zin ontent y flme tomi sorption spetrometry (FAAS). Zin onentrtion in soil ws etermine with the use of Metrnl Qulity Control Mteril (QCM). The limit of etetion for FAAS ws 96 µg m. The totl zin ontent in soil ws etermine with the use of Atomi Asorption Spetrometr AAS, n ioville zin ontent Atomi Asorption Spetrometr GBC 9 AA. The results were nlyze sttistilly in the Sttisti progrm (SttSoft In. 4). Homogeneous groups were ientifie in Tukey s test t signifine level of p =.. The proportion of vrine ssoite with the oserve effets (η ) ws etermine, prinipl omponent nlysis (PCA) ws performe, n the etween the egree of soil ontmintion with zin, enzyme tivity, miroil ounts n physiohemil properties of soil were ompute. The formul propose y Kzyńsk et l. (5) ws use to lulte the iostimultion inex (IF ): IF = A /A, where: A enzyme tivity or miroil ounts in soil with ure, A enzyme tivity or miroil ounts in soil without ure.

3 48 Bull Environ Contm Toxiol (7) 98:46 4 Results n Disussion In this experiment, enzyme tivity ws influene minly y the pplie ose of zin (p.) (Tle ). Urese tivity ws most (46.67%) influene y n intertion etween two experimentl ftors (zin ose x soil inution time) (p.). Ure influene i phosphtse, wheres inution time signifintly ffete the tivity of β-gluosise n, to lesser extent, the tivity of lkline phosphtse. The istriution of vetors esriing enzyme tivity (PCA) revele signifint orreltions etween enzyme Tle Proportion of vrine η oserve in soil ontminte with zin Vriles P Pl Ure Glu Dose of Zn Dose of N Inution time Dose of Zn ose of N Dose of Zn inution time Dose of N inution time Dose of Zn ose of N inution time Error Zn zin, N nitrogen, P i phosphtse, Pl lkline phosphtse, Ure urese, Glu β-gluosise tivity n the pplie ose of zin (Fig. ). Zin oses of 5 n 5 mg Zn + kg stimulte enzyme tivity, wheres higher oses of this element h n inhiitory effet on soil enzymes. This is illustrte y the istriution of ses on plne. Zin s inhiitory influene on the iohemil tivity of soil ws miguous. In stuy y Yng et l. (6), zin oses of to 8 mg kg le to liner reution in urese tivity n n inrese in lkline phosphtse tivity. In the work of Kuhrski et l. (), the negtive onsequenes of zin ontmintion were exerte with n inrese in ose, n zin s inhiitory effet persiste for ys. Enzymes were rrnge in the following orer se on their resistne to zin: β-gluosise > urese > i phosphtse. A similr reltionship ws note y Borowik et l. (4) where zin ose of 4 mg Zn + kg inhiite enzyme tivity in the following orer: lkline phosphtse > urese > i phosphtse > β-gluosise. In the present stuy, the nlyze enzymes were rrnge in the following orer se on their sensitivity to zin ose of 5 mg Zn + kg : urese > i phosphtse > lkline phosphtse > β-gluosise. β-gluosise ws lest suseptile to ll oses of zin, ut oring to Kuhrski et l. (), the enzyme s resistne ereses uner exposure to higher soil iity. Ai phosphtse, lkline phosphtse n β-gluosise respone similrly to soil ontmintion with zin, whih is emonstrte y the proximity of Fig. Hyrolse tivity in soil ontminte with zin. P i phosphtse, Pl lkline phosphtse, Ure urese, Glu β-gluosise; ose of Zn + (mg kg ): I, II 5, III 5, IV 75, V, VI 5; inution time: weeks, weeks; ose of N (mg kg ):, 5, 5 - Ure II- VI- I- V- I- V- VI- PCA :.6% - II- III- Pl III- Glu II- P II- IV- IV- III- IV- IV- V- VI- II- VI- V- I- I- III- IV- IV- III- I- I- V- III- II- V- VI- VI PCA : 4.9%

4 Bull Environ Contm Toxiol (7) 98: vetors representing the nlyze enzymes. Urese ws lso sensitive to zin, ut its response to this hevy metl ws somewht ifferent. This is illustrte y the position of the urese vetor reltive to ses representing the highest zin oses. The neutrlizing effets of nitrogen fertilizer on the iohemil tivity of zin- ontminte soil were oserve fter weeks of inution. Neutrlizing effets were not note fter weeks of soil inution. Our finings suggest tht enzyme tivity is influene y environmentl n nthropogeni ftors n y the enzyme s speifi properties. The zin ontent of soil, etermine y FAAS, inrese linerly. Totl zin onentrtions rnge from mg kg DM soil in unontminte tretments to mg kg DM soil in the most ontminte tretments. The respetive vlues for ioville zin were 8. 4 mg kg DM soil. Similrly to the nominl zin ontent, the ove results orrelte with the miroiologil, iohemil n physiohemil properties of soil. Liner orreltion oeffiients revele signifint negtive orreltions etween Zn + vs. i phosphtse tivity (p.), β-gluosise tivity (p =.4), ounts of opiotrophi (p =.4) n oligotrophi teri (p.) n Atinomyetes ounts (p =.4) (Tle ). There ws highly signifint positive orreltion etween zin ose vs. urese tivity (p.) n fungl ounts (p.). Soil retion signifintly influenes the iohemil properties of soil ontminte with hevy metls, n the introution of ure to the soil eosystem n erese soil ph. The ove ws lso oserve in this stuy. The introution of ure to soil not ontminte with zin le to erese in soil ph (Tle ). The ove reltions n e ttriute to the nitrifition of mmonium ions proue uring ure hyrolysis, whih hnges the soluility of hevy metls. Chnges in soil ph n lso ompromise miroil growth (Geisseler n Sow 4). Zin ontmintion of soil fertilize n not fertilize with ure le to hnges in soil ph. In tretments without ure, soil iity inrese with n inrese in zin ose. In tretments fertilize with nitrogen, zin limite soil iity. The ove n proly e ttriute to zin s inhiitory influene on nitrifition. In stuy y Borowik et l. (4), zin ontmintion of soil limite the evelopment of nitrifying teri n isrupte nitrifition. The nitrifition proess ws effetively inhiite lrey y ose of mg Zn + kg DM soil. In this stuy, the pplition of ure fertilizer lowere soil ph n inrese hyrolyti iity whih ws Tle Soil ph fter weeks of inution Dose of zin (mg Zn + kg ) Dose of nitrogen (mg N kg ) ± ±. ef ±.58 f ±. 4.9 ±. ef 4.8 ±. ef ± ±.9 e 4.97 ±.9 ef ±.9 5. ± ±.9 e 5.4 ± ± ± ±. 5. ±. 5. ±. Homogeneous groups ( f) were rete with the use of ANOVA n Tukey s rnge test t p =.; ±stnr evition Tle Correltion oeffiients emonstrting the strength of reltionships etween the physiohemil properties of soil vs. enzyme tivity n miroil ounts fter weeks of inution Zn T Zn B P Pl Ure Glu Org Cop Olig At Fun P.6**.6** Pl.5.5.* Ure.5**.5**..6 Glu.7**.7**.7**.64**. Org **.4**.5** Cop.7*.8*.6.6**..4*. Olig.57**.58**.9*.44**..8**.4**.68** At.8**.9**.84**.4*..74**...7 Fun.85**.85**.55**..55** **.* ph...55**...9**..*.6**.6**.7 HAC.*.*.4**.*.4**.46**.4.9*.8*.64**.4* TEB.*.*.4.6**.7**.5**.5**..4.5**.* CEC.7*.7*.7.5**.74**.48**.55**.7..46**.* BS.4*.4*.6.44**.68**.55**.49**.7.5.5**.4** Zn T totl zin ontent, Zn B ioville zin ontent, P i phosphtse, Pl lkline phosphtse, Ure urese, Glu β-gluosise, Org orgnotrophi teri, Cop opiotrophi teri, Olig oligotrophi teri, At tinomyetes, Fun fungi, HAC hyrolyti iity, TEB totl exhngele ses, CEC tion exhnge pity, BS se sturtion (*p =.5, **p =.; n = 5)

5 4 Bull Environ Contm Toxiol (7) 98:46 4 A Org Cop Olig At Fun B 6 5 P Pl Ure Glu I F I F Dose of zin (mg Zn + kg - DM soil) Dose of zin (mg Zn + kg - DM soil) I F Org Cop Olig At Fun I F P Pl Ure Glu Dose of zin (mg Zn + kg - DM soil) Dose of zin (mg Zn + kg - DM soil) Fig. Influene of zin on the iostimultion inex (IF ) of miroil growth n enzyme tivity in soil fertilize with ure (A 5 mg N kg ; B 5 mg N kg ). The results were expresse s mens of the vlues note on two nlytil tes ( n weeks of inution). IF = nitrogen hs no effet on miroil growth or hyrolse tivity, IF < nitrogen inhiits miroil growth n hyrolse tivity, IF > nitrogen stimultes miroil growth n hyrolse tivity. Homogeneous groups ( ) were rete seprtely for miroorgnisms, enzymes n nitrogen oses (ANOVA n Tukey s rnge test t p =.). Zn + ose of zin, Org orgnotrophi teri, Cop opiotrophi teri, Olig oligotrophi teri, At tinomyetes, Fun fungi, P i phosphtse, Pl lkline phosphtse, Ure urese, Glu β-gluosise signifintly negtively orrelte with the tivity of ll teste enzymes (P, Ure, Glu: p.; Pl: p =.) n the ounts of the exmine miroil groups (Cop: p =.; Olig: p =.8; At: p.; Fun: p =.), exluing orgnotrophi teri (p =.85). Nitrogen ompouns present in soil re trnsforme minly y miroorgnisms. The ontent of C n the C:N rtio influene miroil growth (Booth et l. 5). Tylor et l. () revele strong orreltions etween orgni mtter ontent, miroil ounts n enzyme tivity. Aoring to Moreno et l. (9), the tivity of urese, i phosphtse n β-gluosise in zin-ontminte soil erese with n inrese in zin ose, ut zin stimulte enzyme tivity in soils with high ontent of orgni mtter. Those oservtions n e lrgely ttriute to the protetive role of orgni mtter in stilizing the iohemil properties of soil (Wyszkowsk et l. 5). In the present stuy, the effets of ure on the iohemil prmeters of soil were evlute in view of nitrogen s ility to neutrlize zin in soils with nrrow C:N rtio. Nitrogen s ility to moify the struture of miroil ommunities in soil hs een wiely isusse in the literture (Strk et l. 7; Wng et l. 5; Yu et l. 5). The vlues of the iostimultion inex (Fig. ) suggest tht nitrogen exerts protetive effets on miroorgnisms in soil ontminte with zin. All miroil groups proliferte t higher rte in soil fertilize with ure. The pplie oses of zin n nitrogen lso plye n importnt role. Zin h stronger stimultory effet on miroil growth in tretments expose to the highest ose of ure thn in tretments fertilize with 5 mg N kg. Our finings inite tht nitrogen exerts protetive effet on miroil

6 Bull Environ Contm Toxiol (7) 98:46 4 prolifertion in zin-ontminte soil, whih influenes the tivity of soil enzymes. The enzyme iostimultion inex provies informtion out ure s neutrlizing effets on the tivity of lkline phosphtse, urese n β-gluosise in zin-ontminte soil. The egree of enzyme stimultion ws etermine y the nitrogen ose n zin onentrtions in soil, whih ws prtiulrly notiele in urese tivity in tretments expose to the highest zin oses ( n 5 mg Zn + kg soil DM). Other uthors lso emonstrte tht nitrogen fertiliztion inrese the tivity of β-gluosise (Siy-Cork et l. ), urese n i phosphtse (Siy-Cork et l. ; Wng et l. 8). Nitrogen stimultes soil miroorgnisms whih proue more soil enzymes when iogeni elements eome more ville. In stuy y Jio et l. (), C, N n P onentrtions in soil were orrelte with phosphtse tivity, ut not with urese tivity. The ove results inite tht enzymes respon ifferently to vrious soil iohemil prmeters. The results of this n other stuies suggest tht enzyme tivity is onitione y vrious overlpping ftors, inluing physiohemil prmeters, exposure to toxi ompouns n the influene of stimulting n protetive sustnes. In this experiment, zin h stimultory effet when pplie in smller oses, ut higher oses of this element strongly inhiite the tivity of soil enzymes. Nitrogen s influene on enzyme tivity ws lso etermine y the pplie ose of ure. The protetive effet exerte y nitrogen on enzymes vrie sujet to hnges in soil iity. The responses of the nlyze hyrolses to ifferent oses of zin n nitrogen vrie over time. Aknowlegements This reserh stuy ws supporte y the Polish Ministry of Siene n Higher Eution. Open Aess This rtile is istriute uner the terms of the Cretive Commons Attriution 4. 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