Remediation of copper contaminated soil by using different particle sizes of apatite: a field experiment

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1 DOI /s y RESEARCH Open Access Remedition of copper contminted soil y using different prticle sizes of ptite: field experiment Jinfeng Xing 1,2, Tintin Hu 1, Long Cng 1* nd Dongmei Zhou 1 *Correspondence: cnglong@isss.c.cn 1 Key Lortory of Soil Environment nd Pollution Remedition, Institute of Soil Science, Chinese Acdemy of Sciences, Nnjing , Chin Full list of uthor informtion is ville t the end of the rticle Astrct The prticle size of ptite is one of the criticl fctors tht influence the dsorption of hevy metls on ptite in the remedition of hevy metl contminted soils using ptite. However, little reserch hs een done evluting the impct of different prticle sizes of ptite on immoiliztion remedition of hevy metl polluted soils in field. In this study, the dsorption isotherml experiments of copper on three kinds of ptite ws tested, nd the field experiment y using different prticle sizes ptite [nnohydroxyptite (NAP), micro-hydroxyptite (MAP), ordinry prticle ptite (OAP)] t sme dosge of 25.8 t/h (1.16 %, W/W) ws lso conducted. Ryegrss ws chosen s the test plnt. The ryegrss iomss, the copper contents in ryegrss nd the copper frctiontions in soil were determined fter field experiments. Results of dsorption experiments showed tht the dsorption mounts of copper on OAP ws the lowest mong different prticles. The dsorption mounts of copper on MAP ws higher thn NAP t high copper equilirium concentrtion (>1 mmol L 1 ), n opposite trend ws otined t low copper concentrtion (<1 mmol L 1 ). In the field experiment, we found tht the ppliction of different ptites could effectively increse the soil ph, decrese the ville copper concentrtion in soil, provide more nutrient phosphte nd promote the growth of ryegrss. The ryegrss iomss nd the copper ccumultion in ryegrss were the highest in MAP mong ll tretments. The effective order of ptite in phytoremedition of copper contminted field soil ws MAP > NAP > OAP, which ws ttriuted to the high dsorption cpcity of copper nd the strong relesing of phosphte y MAP. Keywords: Aptite, Prticle size, Copper, Adsorption, Field experiment Bckground Soil contmintion is n excess of ny compound or element, which results in serious impct on ecosystems, groundwter, griculturl productivity nd humn helth (Adrino 2001). Copper is n essentil element: it forms orgnic complexes nd metlloproteins, especilly hemogloin (Kos nd Lestn 2004). But high concentrtion of Cu could cuse serious hrms to iot nd humn (Adrees et l. 2015). Therefore, effective mendment nd proper tretment to decrese its vilility nd moility ecome necessry. Aptite is common nme for different minerls with the formul X 5 (YO 4 ) 3 (OH, F, Cl), where X is divlent metl, such s C, B, P, etc., nd Y is phosphorous, possile replced 2016 The Author(s). This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde.

2 Pge 2 of 16 y other elements such s As, V, C (Nrsrju nd Phee 1996). Studies focusing on immoiliztion of hevy metls y ptite such s nturl minerl phosphte nd synthetic ptite hve een extensively reported (Lperche et l. 1996; M et l. 1995; Miretzky nd Fernndez-Cirelli 2008). Hydroxyptite (HAP) is memer of ptite minerl fmily, with the formul C 10 (PO 4 ) 6 (OH) 2, nd hs high dsorption cpcity for divlent hevy metl ions nd hs een used for wstewter tretment (Cormi et l. 2008; M et l. 1994; Sndrine et l. 2007; Smicikls et l. 2006) nd soil remedition (Chturvedi et l. 2006; Keller et l. 2005). Metl ions such s P(II), Cu(II), Co(II), Zn(II), nd Cd(II) re successfully immoilized y dsorption nd precipittion on the surfce of HAP (Cormi et l. 2007; Lusvrdi et l. 2002; Sndrine et l. 2007; Smicikls et l. 2006). Moreover, Chen et l. (2006) indicted tht the soluility nd grin size of rock phosphte could ffect the effectiveness of the mendment in in situ remedition technology, nd they concluded tht the rock phosphte with smll grin size (<35 μm) ws superior to grin sizes lrger thn 35 μm t reducing hevy metls uptke in plnt. Sugiym et l. (2002) found tht the exchnge cpcity of ptite of prticle size under 0.85 mm to P(II) is much etter thn tht of prticle size from 0.85 to 1.70 mm. In prticulr, nno-mterils represent promising ppliction in some res due to their high surfce res, smll sizes nd specil chemicl rection (Zhng et l. 2010). Wng et l. (2009) found tht nnoscle HAP exhiits strong dsorption for Cu, nd is good dsorent for immoiliztion of hevy metls. Liu nd Zho (2007) found nno-prticles hd etter performnce thn norml prticles during studying the immoiliztion of Cu(II) in soil using iron phosphte. Although the immoiliztion of hevy metls using ptite hs een proved successfully in the lortory, the remedition of hevy metl contminted soils y nnohydroxyptite (NAP), micro-hydroxyptite (MAP) nd ordinry prticle size of ptite (OAP) in the field is very limited. Thus, the ojectives of this study were to evlute the Cu dsorption cpcity on different prticle size of ptite (NAP, MAP, OAP) nd the remedition ility of Cu contminted soil y different ptites in the field condition. Methods Soil The test site is locted in Guixi city, Jingxi province, Chin ( E, N). This re is influenced y Southest Asi Monsoon, so the climte is wrm nd humid. The soil ws polluted y the sewge nd wste gs from Cu smelting fctory. The test field hs een ndoned for mny yers nd there re mny serious phenomen such s desertifiction, cidifiction nd hevy mentl pollution. The soil ph, orgnic mtter content, ville N, P nd K concentrtion were 4.25, 30.8 g kg 1, 46.7, 122 nd 41.5 mg kg 1, respectively. The totl Cu, Zn, P nd Cd concentrtions were 580, 85.5, 37.6 nd 0.35 mg kg 1, respectively. Mterils Nno-hydroxyptite (NAP), micro-hydroxyptite (MAP) nd ordinry prticle size of ptite (OAP) were used s dsorption mterils nd purchsed from Nnjing emperor nno mteril Co. Ltd. nd Xinti phosphorus chemicl plnt. The sic physicochemicl properties nd the TEM imging of ptites re shown in Tle 1 nd Fig. 1. Ryegrss (Lolium perenne L.), supplied y Nnjing Shenzhou Seeds Industry Co., Ltd., ws chose s the test plnt.

3 Pge 3 of 16 Tle 1 The sic properties of the different ptites Mteril Prticle Purity (%) Shpe BET (m 2 g 1 ) ph Zet potentil (mv) P (mg kg 1 ) Cu (mg kg 1 ) Zn (mg kg 1 ) Cd (mg kg 1 ) C/P NAP 60 nm >96 Aciculr MAP 12 μm >96 Sphere OAP 0.15 mm >96 Bulk

4 Pge 4 of 16 Fig. 1 The TEM imges of different ptites. nno-hydroxyptite (NAP), micro-hydroxyptite (MAP), c ordinry prticle size of ptite (OAP) Adsorption isotherml experiments The dsorption isotherml experiments were crried out in 50 ml polypropylene centrifugtion tues y mixing 0.05 g of different ptites with 25 ml 0.01 mol L 1 NNO 3 contining 25 mmol L 1 MES (4-Morpholineethnesulfonic cid hydrte). The

5 Pge 5 of 16 ph of suspensions were djusted to 5.5 y diluted HCl or NOH. Afterwrds, 5 ml of 0.01 mol L 1 NNO 3 with different concentrtion of Cu(NO 3 ) 2 were dded into the tues. The initil concentrtion of copper in the solutions were 0, 0.2, 0.4, 1.0, 1.6, 2.0, 5.0 nd 10.0 mmol L 1, respectively, nd ech tretment ws duplicted. All smples were shken for 24 h t 25 C, followed y centrifuging t 9000 rpm for 15 min. The superntnts were filtered through 0.22 μm Millipore filter. The Cu concentrtions in the filtrte were mesured y tomic sorption spectroscopy (AAS). Adsored copper ws quntified y the difference etween initil nd finl copper concentrtions in solution fter control correction. The dsorption isotherms were fitted y the Lngmuir nd Freundlich equtions (Eqs. 1 nd 2, respectively). Q e = Q m K L C e /(1 + K L C e ) Q e = K f Cn e (1) (2) where C e (mmol L 1 ) is the equilirium concentrtion of copper in solution, Q m (mmol kg 1 ) is the dsored quntity of copper, Q m (mmol kg 1 ) nd K L (L kg 1 ) indicte the mximl monolyer dsorption cpcity nd constnt relted to dsorption energy, respectively. K f nd n re Freundlich constnts giving n estimtion of the dsorption cpcity nd intensity, respectively. Field experiment The field plot trils were conducted to investigte the remedition effect y using different prticle size of ptite in the Cu contminted field. Triplicted tril plots (2 m 2 m) were lid out y rndomized lock design. The tril plots were seprted y 0.5 m high PVC sheets with 0.2 m ove ground nd 0.3 m underground. The ppliction dosges of the ptite were 1.16 % (W/W) of the topsoil (0 20 cm) weight (25.8 ton h 1 ). The tretments were leled s NAP, MAP, OAP nd CK (without ny mendment), respectively. On Novemer 13, 2010, the site ws ploughed nd hrrowed. In order to void the suspension nd gglomertion of ptites (especilly NAP), the ptite powders were spred on the soil surfce nd mixed evenly with topsoil, then ech plot ws irrigted with 100 L of tp wter. In ech plot, 20 g of ryegrss seeds ws sown nd 0.5 kg of compound fertilizers (the totl content of N, P 2 O 5 nd K 2 O were 15 %) ws pplied. After 18 weeks (the first hrvest, Mrch 29, 2011) nd 21 weeks (the second hrvest, April 22, 2011) growing, the oveground iomss of ryegrss ws hrvested, nd then 0.04 kg of the ure ws pplied per plot. After 23 weeks of growth (the third hrvest, My 8, 2011), the shoot nd root of ryegrss were hrvested, nd the soil smples were lso collected. The shoot smples ws thoroughly wshed y tp wter nd rinsed y deionized wter. The root smples were thoroughly wshed using 2-step wshing progress, which consisted of first wsh with EDTA (20 mmol L 1 ) nd second rinse with deionized wter. Both shoot nd root smples were preliminrily dried t 105 C for 30 min, nd then switched to 70 C until constnt weight hs een reched (out 48 h). The dry mtters were grounded to fine powders using mechnicl grinder fter the yields were recorded. The soil smples were ir dried nd sieved through 2 nd mm sieves efore mesurements.

6 Pge 6 of 16 Anlyticl methods The physicl structures of different ptites were imged y Trnsmission Electron Microscope (TEM, JEOL TEM-2100,Jpn). The minerl phses were nlyzed using X-ry Diffrction Spectrometer (XRD, Rigku Ultim IV, Jpn) with the diffrction pttern compred to the referenced XRD stndrd mp (Joint Committee on Powder Diffrction nd Stndrds, NO ). The FTIR spectr ws record on Nicolet 380 ptterns (USA) of the solid were tken (scn in cm 1 ), s out 2 mg smples were suspended in out 150 mg of KBr. Zet potentil ws determined y Nno- Brook 90Plus PLAS (Brookhven Instruments, Americ). The specific surfce re ws mesured y the Brunuer-Emmett-Teller (BET) method using the specific surfce re utomtic nlyzer (Quntchrome Autosor-iQ, Americ). The ph of ptites were mesured in suspension with the cron dioxide-free distilled wter to solid rtio of 1:6 (W/V) y using ph meter (phs-2b, Shnghi Rex Electric Chemicl Co. Ltd., Chin). The ptites were digested using HNO 3 -HF-HClO 4 (10:10:1, V/V) mixture nd the digestion solutions were nlyzed to quntify the concentrtions of Cu, Zn, P, Cd, C nd P in ptites using flme tomic sorption spectrophotometer (Hitchi Z-2000, Jpn). Soil ph, orgnic mtter content nd ville phosphorus contents were nlyzed sed on the method given y Lu (2000). The soil ph were mesured in suspension with the cron dioxide-free distilled wter to soil rtio of 2.5 (V/W) y ph meter (phs-2b). Soil orgnic mtter content ws nlyzed y dichromte oxidtion method. Aville soil phosphorus ws extrcted y the mixed solution contined 0.03 mol L 1 NH 4 F nd mol L 1 HCl nd determined y ICP (PerkinElmer Optim 8000). To determine the totl concentrtion of metls in soil, the soil smples were pssed through mm pore-size sieve nd dried t 105 C for 4 h, nd then digested in n HNO 3 -HF-HClO 4 (10:10:1, V/V) mixture. Blnk nd soil stndrd mterils (GBW nd GBW-07406) (Chin Ntionl Center for Stndrd Mteril) were used for qulity control. The Cu frctions were identified y the BCR sequentil extrction method from the Europen Community Bureu of Reference (Ruret et l. 2000), nd the cid solule, reducile, oxidizle, nd reducile frctions of Cu were extrcted y 0.11 mol L 1 cetic cid, 0.1 mol L 1 hydroxylmine hydrochloride (ph 2), 30 % W/V H 2 O 2 nd 1 mol L 1 NH 4 OAc (ph 2), HNO 3 HF HClO 4 (1:2:1, V/V), respectively. The recovery of totl Cu concentrtion ws controlled in %. TCLP (Toxicity Chrcteristic Leching Procedure) method ws used to estimte the vrition of Cu toxicity in mended soils. This procedure diluted 5.7 ml glcil cetic cid in 1 L deionized wter nd used 20 ml of this solution (ph 2.88) to extrct copper from the 1 g of soil smples (Sun et l. 2006). For the Cu concentrtion in ryegrss shoots nd roots, out 0.5 g smple ws weighed in 50 ml tringulr flsk with mixture of HNO 3 (20 ml) nd H 2 O 2 (4 ml) nd left t room temperture overnight. Then, the smples were digested on n electric heting plte ( C). Blnk nd stndrd reference mterils (GBW nd GBW ) (spinch leves nd rice grin, Chin Ntionl Center for Stndrd Mteril) were used for qulity control in the digestion nd nlysis processes. The Cu concentrtions of in digestion solutions were nlyzed using flme tomic sorption spectrophotometer (Hitchi Z-2000, Jpn).

7 Pge 7 of 16 Sttisticl nlysis SPSS 18.0 nd Excel 2003 were used for sttisticl nlysis. Lngmuir nd Freundlich equtions were pplied to fit the copper dsorption isothermls. The results of field experiments were sujected to nlysis y one-wy nlysis of vrince (ANOVA) followed y Duncn multiple comprisons (p < 0.5) using SPSS Results nd discussion Chrcteristics of different ptites TEM imges (Fig. 1) showed tht the shpes of NAP, MAP nd OAP were ciculr, sphere nd ulk, respectively. The order of prticle size of different ptite ws NAP < MAP < OAP, which ws on the opposite trend with the specific surfce re y the BET method. The results of XRD nlysis showed NAP nd MAP consisted with the stndrd crd of hydroxyptite (JCPDS 9-432), nd fluorptite (65 %) ws found s the primrily minerl in OAP, nd the second most undnt minerl ws dolomite (30 %), followed y hydromic (2 %), tlcum (2 %) nd kolinite (1 %) (Fig. 2). The XRD nd FTIR spectrogrms (Figs. 2, 3) indicted the OAP is not pure ptite. Some other minerls (dolomite, hydromic, etc.) didn t contin phosphorus, which decresed the phosphorus content in OAP nd incresed the C/P rtio of OAP to The C/P molr rtio of NAP nd MAP were 1.70 nd 1.61, respectively, which is close to the idel rtio of Fluorptite Dolomite OAP Intensity MAP Hydroxylptite Hydroxylptite NAP Thet Fig. 2 X-ry diffrction (XRD) pttern of different ptites

8 Pge 8 of 16 OAP Trnsmission (%) MAP NAP Wve numer (cm -1 ) Fig. 3 Fourier trnsform infrred spectroscopy (FTIR) spectr of different ptites Zet potentil of MAP ws 44.4 mv nd higher thn NAP nd OAP (solute vlue), which indicted tht MAP preferentilly disperses in soil nd mixes with soil components compre to NAP nd OAP. The concentrtions of hevy metl in ptite were mg kg 1 for Cu, mg kg 1 for Zn, mg kg 1 for P nd mg kg 1 for Cd, respectively, nd slightly higher thn the concentrtion previously reported (Cui et l. 2013, 2014). Adsorption of Cu on different ptites Adsorption isotherms of Cu on ptites t ph 5.5 re given in Fig. 4. The dsorption of Cu on ptite grdully incresed with the increse of copper concentrtion in the equilirium solution, which is in greement with those reported y Wng et l. (2009) nd Cui et l. (2014). Figure 4 show the dsorption of Cu in mmol kg 1. In cse of lower Cu concentrtion (elow 1.0 mmol L 1 ) in equilirium solution, the dsorption trend (from high to low cpcity) of ptites ws NAP > MAP > OAP. The dsorption trend ws similr to the study given y M et l. (1995), in which it hs een suggested tht for phosphte rock, higher specific surfce re incresed its dissolution rte nd promoted the dsorption nd immoiliztion of hevy metls. The dsorptions of Cu were showed in mmol m 2 in Fig. 4. The Cu dsorption on NAP nd MAP were similr with the result in Fig. 4. Tht suggested tht NAP displyed higher immoilizing effect thn MAP due to its high surfce re (Tle 1). However, there ws different etween Fig. 4, for the Cu dsorption cpcity on OAP, which indicted the composition nd purity of OAP ffected the Cu dsorption on OAP. However, n interesting phenomenon ws oserved tht the Cu dsorption cpcity on MAP incresed rpidly nd ws higher thn tht on NAP when the equilirium concentrtion of Cu exceeded 1.0 mmol L 1 (Fig. 4). The highest Cu dsorption cpcity on NAP nd MAP were 800, 2300 mmol kg 1, respectively, nd the dsorption cpcity on OAP kept stying t very low level (elow 214 mmol kg 1 ). The prmeters for Lngmuir nd Freundlich fitting re listed in Tle 2, the dsorption isotherms of MAP nd OAP in 0 10 mmol L 1 were not fitted y Lngmuir nd Freundlich models nd OAP in 0 1 mmol L 1 ws not fitted y Lngmuir model. Cheung et l. (2002) found tht

9 Pge 9 of 16 Adsorption of Cu (mmol kg -1 ) Adsorption of Cu (mmol m -2 ) NAP MAP OAP NAP MAP OAP Cu in equilirium solution (mmol L -1 ) Cu in equilirium solution (mmol L -1 ) Fig. 4 Adsorption isotherms of Cu on the three kinds of ptite t ph 5.5 (The unit of Cu sorption mount in y-xis were expressed y mmol kg -1 () nd mmol m -2 (), respectively) Tle 2 Lngmuir nd Freundlich model prmeters of different ptite t ph 5.5 Ce (mmol L 1 ) Mteril Lngmuir Freundlich K L Q m R 2 P K F 1/n R 2 P 0 10 NAP < < NAP < < MAP < < OAP <0.05 The dsorption isotherms of MAP nd OAP in 0 10 mmol L 1 were not fitted y Lngmuir nd Freundlich models nd OAP in 0 1 mmol L 1 were not fitted y Lngmuir models, so their model prmeters re not listed Lngmuir eqution cn used to descrie the sorption of Cu y one chr ( μm prticle size) minly composed with hydroxyptite. This difference might e due to tht the different composition nd property of sorent nd experiment conditions. The dsorption isotherms of NAP were well fitted y Lngmuir nd Freundlich models, nd the correltion coefficient (R 2 ) were 0.96 (0 10 mmol L 1 ) nd 0.98 (0 1 mmol L 1 ) in Lngmuir models nd 0.95 (0 10 mmol L 1 ) nd 0.94 (0 1 mmol L 1 ) in Freundlich models, respectively. These results showed tht the Lngmuir models is etter in descriing the Cu dsorption ehvior on NAP nd the dsorption ehvior ws minly monomoleculr dsorption process, similr to studies given y Chen et l. (2010) nd Wng et l. (2009). Adsorption rection of Cu on MAP is divided into two phses (Fig. 2): first, the sorption of Cu on MAP corresponds siclly to Lngmuir eqution t

10 Pge 10 of 16 low Cu equilirium concentrtion (<1.0 mmol L 1 ); second, the dsorption ecme liner with oth rte nd cpcity incresed linerly t high Cu equilirium concentrtion (>1.0 mmol L 1 ). These indicted tht the dsorption of Cu on MAP might e multilyer dsorption nd the surfce of MAP hd strong ffinity of Cu t higher Cu concentrtion. The est metl sorption on ptites need poor crystllinity (Chen et l. 1997, ). XRD nlysis showed the crystllinity of MAP ws the poorest, so the Cu dsorption cpcity on MAP ws the est. Effect of different ptites on soil ph, TCLP extrcted Cu concentrtions The ddition of NAP, MAP nd OAP led to the increse of soil ph (Fig. 5A), which is similr with previous reports (Chlopeck nd Adrino 1996; M et l. 1995) nd proly due to the hydroxyl, clcium nd some other sic groups of ptite (Boisson et l. 1999; Knox et l. 2003). However, the soil ph of different tretments significntly incresed in Mrch 2011, nd then decrese of soil ph ws oserved in My Similrly, Cui et l. (2014) found tht with the ppliction of ptite, soil ph significntly incresed fter 1 yer nd decresed fter 4 yers. They speculted tht it might e ttriuted to the fct tht test site ws locted in n cid deposition zone. As shown in Fig. 5A, the soil ph followed the sme order of OAP > MAP > NAP > CK in Mrch 2011 nd My 2011, which ws consisted with the ph order of ptites (OAP > MAP > NAP). Moreover, the soil ph treted y NAP slightly chnged wheres significnt ph increses A 8.0 ph initil 03/ / B 250 TCLP extrcted Cu concentrtion (mg kg -1 ) initil 03/ / CK NAP MAP OAP Tretment Fig. 5 Effect of different ptites on soil ph (A) nd TCLP extrcted Cu concentrtions (B) (different letters indicted significnt differences etween tretments t the sme time (p < 0.05)

11 Pge 11 of 16 were identified in soil treted y OAP nd MAP. This indicted tht OAP nd MAP is more effective in incresing soil ph thn NAP under the identicl condition. Figure 5B shows the concentrtion chnges of TCLP extrcted Cu cused y the ddition of NAP, MAP nd OAP, nd the concentrtion incresed in Mrch 2011 compred to Novemer Possily due to the open experimentl system in field, the immoiliztion process of Cu ws reltively slow. But fter two months since Mrch 2011, TCLP extrcted Cu decresed significntly lone with the order of OAP > CK > MAP > NAP. The results indicted tht NAP nd MAP were le to immoilize TCLP extrcted Cu nd NAP ws more effective thn MAP. However the soil ph in MAP tretment were higher thn those in NAP tretment, especilly there ws significnt differences etween NAP nd MAP in Mrch 2011 (Fig. 5A). The immoiliztion of Cu y HAP ws possily more dependent on the high dsorption quntity of Cu rther thn the soil ph compring to tht y MAP. Nevertheless, more experiments re still needed to further study the effect of soil ph on the immoiliztion mechnism of Cu y ptite. Effect of different ptites on the Cu specition in soil The moility of hevy metls, their iovilility nd relted eco-toxicity to plnt, depend strongly on their specific chemicl frctions (Chen et l. 2014; Wng et l. 2012). Appliction of mendment cn chnge the chemicl frctions of hevy metls in soil nd reduce their toxicity (Mckie et l. 2015; Montenegro et l. 2015; Rodriguez-Vil et l. 2015). The concentrtion of Cu nd the percentges of four frctions in soil estimted y BCR method re shown in Tle 3. In the untreted soil (CK), the cid solule frction (250 mg/kg, 47.7 %) ws predominnt followed y reducile frction (134 mg/kg, 25.5 %) nd oxidizle frction (109 mg/kg, 20.7 %), the lowest percentge ws found in residul frction (31.9 mg/kg, 6.08 %). After ppliction of NAP, MAP nd OAP, the Cu concentrtion in cid solule frction decresed significntly from 47.7 % (CK) to 31.2 % (NAP), 39.6 % (MAP) nd 40.5 % (OAP), respectively. By contrst, the Cu concentrtion in reducile frction incresed significntly with the highest incresing in the NAP tretment nd the lowest incresing in the OAP tretment. Only smll chnges in residul frction were identified mong ll tretments. The results indicted tht the ppliction of ptites reduced the vilility of Cu nd ttenuted its risk y decresing the high toxic nd moile cid solule frction nd menwhile immoilizing Cu to reltively stle reducile nd oxidizle frction. The order of immoilizing is: NAP > MAP > OAP, ut there hve no significnt difference in the cid solule frction etween NAP nd Tle 3 Effect of different ptites on the Cu frctiontions in soil Tretment F1 F2 F3 F4 Recovery % mg kg 1 % mg kg 1 % mg kg 1 % mg kg 1 % CK c c NAP MAP c c OAP c F1, cid solule frction; F2, reducile frction; F3, oxidizle frction; F4, residul frction; Men vlues followed y different lowercse letters in ech column mens significntly different (p < 0.05, n = 3)

12 Pge 12 of 16 MAP. A previous study (Cui et l. 2013) showed tht MAP ws more effective thn NAP in immoilizing Cu under the greenhouse condition. The difference might e due to the physic-chemicl properties of ptites nd experiment conditions (pot or field experiment). The prticle size of NAP (40 nm) nd MAP (3 μm) in Cui s rticle (Cui et l. 2013) is smller thn tht in our study (60 nm NAP nd 12 μm MAP), which could cuse d dispersiility for NAP in soil nd susequently wek the immoiliztion effect. Nevertheless, more studies re needed to verify the different immoiliztion mechnisms of Cu y NAP nd MAP under different conditions. Possile mechnisms of metl retention y ptite included: (1) ion exchnge t the surfce of ptite (Xu et l. 1994); (2) surfce complextion (Co et l. 2004); (3) precipittion of some morphous to poorly crystlline, mixed metl phosphtes; nd (4) sustitution of C in ptite y other metls during recrystlliztion (or coprecipittion) (Chen et l. 1997, ; Xu et l. 1994). According to the report of Lindsy (2001), the structure of hydroxyptite is similr to tht of fluorptite, F occupying the OH sites on the sixfold xis. But hydroxyptite (log K = 14.46) hs higher soluility thn fluorptite (log K = 0.21). M et l. (1995) ttriuted tht the higher P removl efficiency to higher soluility nd purity of hydroxyptite thn phosphte rocks. Drouet (2015) indicted tht Gis free energy lso estimted the soluility of ptites. Moreover, Kludjerovic-Rdoicic nd Ricevic (2010) suggested tht the ΔG 0 of HAP ws greter thn tht for LA (fluorptite), nd HAP showed lrger ffinity for P removl y the greter vlue of the sorption cpcity. In this study, the immoilizing cpcity of Cu followed the order of NAP > MAP > OAP, nd the OAP ws primrily fluorptite nd not pure. Therefore, one possiility reson could e the higher soluility of MAP nd NAP thn OAP. Furthermore, lrger specific surfce res were determined for NAP nd MAP thn OAP (Tle 1), which is likely enhncing the sorption of metls nd leding to the decrese of the iovilility of metl (Chen et l. 2006; Zhng et l. 2010). Thermodynmic properties might e different somewht depending on the grin size of the ptite (Drouet 2015), therefore, more studies out thermodynmic of NAP nd MAP need to conduct. Effect of different ptites on ryegrss iomss Tle 4 shows the shoot nd root iomss of ryegrss in different tretments. Ryegrss ws not le to grow in the CK tretment (without the ddition of mendment), ut ppliction ptites resulted in improved the growth of ryegrss. In generl, the est growth responses (shoot nd root) were oserved t MAP ddition mong ll tretments. And there were significnt differences etween this tretment nd Tle 4 Effect of different ptites on the iomss of ryegrss Tretment Biomss of shoot (g) Biomss of root (g) 1st cutting 2nd cutting 3rd cutting Totl iomss of shoot (g) Totl iomss of ryegrss (g) NAP MAP OAP c c Men vlues followed y different lowercse letters in ech column mens significntly different (p < 0.05, n = 3)

13 Pge 13 of 16 others. The trend of shoot, root nd totl iomss of ryegrss ws MAP > NAP > OAP, lthough there ws no significnt difference etween NAP nd OAP. It is well known tht nutrient vilility is of primry importnce to the productivity. It cn e seen from Fig. 6 tht the ptites significntly incresed the concentrtion of ville phosphorus nd the incresing order ws MAP > NAP > OAP, especilly there hve significnt difference etween MAP nd NAP in My Therefore, the relese of phosphorus y these different ptites ws one of the importnt fctors to the iomss vrition. The Cu dsorption on NAP ws higher thn tht on OAP (Fig. 4), ut no significnt difference of ryegrss iomss ws found etween NAP nd OAP. So nother importnt fctor of ryegrss growth ws the soil ph, in the OAP tretment, significntly higher thn the soil ph ( ) in the NAP tretment (Fig. 5). The neutrl soil ph ws eneficil to the growth of ryegrss (Mor et l. 2005; Ross et l. 2007). Effect of different ptites on the Cu content nd ccumultion in ryegrss Tle 5 shows the Cu content nd ccumultion in ryegrss of different tretments. After pplying ptites with different prticle sizes, the Cu contents in the shoot of different cutting time ws different. The Cu contents in shoot fter the first cutting ws much higher thn those of the second nd the third cutting excepted for the NAP tretment, which my e relted to the dilution effect resulting from the significntly incresing of ryegrss iomss in the second nd the third cutting. There ws no significnt 1400 Avile P concentrtion (mg kg -1 ) initil 03/ /2011 c 0 CK NAP MAP OAP Tretment Fig. 6 Effect of different ptites on ville phosphorus concentrtion [different letters indicted significnt differences etween tretments t the sme time (p < 0.05)] Tle 5 Effect of different ptites on Cu concentrtion nd Cu ccumultion in ryegrss Tretment Cu concentrtion (mg kg 1 ) Cu ccumultion (mg plot 1 ) Shoot Root Shoot Root 1st cutting 2nd cutting 3rd cutting NAP MAP OAP Men vlues followed y different lowercse letters in ech column mens significntly different (p < 0.05, n = 3)

14 Pge 14 of 16 difference of Cu contents in the shoot mong different tretments, except tht the Cu content in the third cutting shoot of the MAP tretment ws significntly less thn those in other tretments. However, the Cu content of root in the MAP ws the highest in ll tretments, nd ws out 1.69, 3.90 times tht in NAP, OAP, respectively. According to the report of Lperche et l. (1997), the tretments with high quntities of phosphorus dded resulted in the content of P in the plnt roots the sme or higher thn the untreted soil. Therefore, we speculted tht the higher Cu content of root in the MAP my e ttriuted to its highest phosphorus concentrtion (Fig. 6). The Cu ccumultion mount in shoot (1143 mg plot 1 ) nd root (5580 mg plot 1 ) in the MAP tretment were 5.16 nd 2.86 times higher thn tht in the NAP tretment, from which it ws concluded tht the ryegrss comined MAP ws more effective thn NAP or OAP in removing Cu from soil. The overll trend of Cu ccumultion ws MAP > NAP > OAP, which ws corresponding to the ryegrss iomss. This field study indicted tht the ryegrss iomss ws the mjor fctor for Cu ccumultion, wheres the different prticle size of ptite hd smll effect on the Cu content in the shoot of ryegrss. Conclusions Our study clerly showed tht ptites effectively incresed the soil ph nd improved the growth of ryegrss, decresed the concentrtion of ville Cu in soil, nd promoted the Cu trnsformtion from ctive to stle frction in field experiment. Although the specific surfce re of NAP ws lrger thn MAP nd OAP, the dsorption of Cu on MAP ws etter thn tht on NAP in high Cu concentrtion (>1 mmol L 1 ) in dsorption experiment. The ryegrss iomss nd the copper ccumultion in ryegrss were the highest in MAP mong ll tretments. The effective order of ptite in phytoremedition of copper contminted field soil ws MAP > NAP > OAP, which ws ttriuted to the high dsorption cpcity of copper nd the stronger relesing of phosphte y MAP. Authors contriutions JX crried out the whole experimentl studies, drfted nd revised the mnuscript. TH prticipted in the field reserch nd helped to nlyze the experimentl results. LC designed the experiments nd conceived of the study, performed the sttisticl nlysis nd drwing. DZ helped to revise the mnuscript. All uthors red nd pproved the finl mnuscript. Author detils 1 Key Lortory of Soil Environment nd Pollution Remedition, Institute of Soil Science, Chinese Acdemy of Sciences, Nnjing , Chin. 2 University of Chinese Acdemy of Sciences, Beijing , Chin. Acknowledgements This reserch ws finncilly supported y the Project of Science nd Technology Service Network Inititive, Chinese Acdemy of Sciences (KFJ-EW-STS-016) nd the Ntionl Nturl Science Foundtion of Chin ( , ). The uthors thnk Dr. Tinrn Sun for his constructive suggestions of revmping the mnuscript. Competing interests The uthors declre tht they hve no competing interests. Received: 21 Jnury 2016 Accepted: 2 June 2016 References Adrees M, Ali S, Rizwn M et l (2015) The effect of excess copper on growth nd physiology of importnt food crops: review. Environ Sci Pollut Res 22(11): Adrino DC (2001) Trce elements in terrestril environments: iogeochemistry, iovilility, nd risks of metls. Springer, New York

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