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2 Chpter 4 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus (Nelumo nucifer) nd Chinese Sge (Slvi miltiorrhiz) Dike Tin, Amei Liu nd Ynci Xing Additionl informtion is ville t the end of the chpter Astrct Appliction of iochr hs mny eneits in griculture, to understnd eneits of iochr in crop production nd remedition of hevy metl pollution, Asin lotus (Nelumo nucifer) s n qutic crop nd Chinese sge (Slvi miltiorrhiz) s trditionl medicinl her were used to evlute iochr s efects on plnt growth nd cdmium (Cd) ccumultion in plnts in the rtiicilly Cd-polluted condition in continers. In oth cses, dding iochr (4% to 32% in soil mix) signiicntly incresed plnt iomss. However, its impct on plnt physiologicl trits were uncler. In Asin lotus, the Cd content in rhizomes, petioles, nd leves signiicntly incresed y 69%, 81% nd 55%, respectively s 32% iochr dded. Menwhile, mximum reduction (71%) showed on ioccumultion coeicient of Cd, nd n up to 1.3 folds increse occurred on Cd trnsfer coeficient of underground to oveground tissues, which indicted tht iochr efectively prevented Cd uptke in mjor edile prts. In Chinese sge, dding 32% iochr signiicntly decresed Cd content in leves nd roots y 52.81% nd 43.63%, respectively. Therefore, s vlule soil mendment of improving plnt growth nd reducing hevy metl uptke, iochr hs huge potentil in green griculture production nd remedition of hevy-metl polluted environment. Keywords: iochr, hevy metl pollution, cdmium, Nelumo nucifer, lotus, Slvi miltiorrhiz, Chinese sge, dnshen, plnt growth, vegetle, medicinl plnt 1. Introduction With the rpid development of urniztion, industry, nd griculture, hevy metl contmintion in wter, soil, nd the tmosphere hs een incresing. Hevy metls ccumulte in 2017 The Author(s). Licensee InTech. This chpter is distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

3 50 Engineering Applictions of Biochr plnts, nimls, nd humn odies, therefore, humn helth is exposed to gret potentil thret through the food chin. Biochr is n emerging environment functionl mteril nd hs een proven to e n eicient dsorent for toxic orgnic pollutnts nd hevy metls [1 4]. With excellent physicochemicl structure, iochr hs gret griculturl eneits nd the potentil for remedition of contminted soils y improving soil physicochemicl properties [5, 6], incresing microil ctivity in soil [7, 8], reducing nutrient leching [9], nd enhncing nutrient vilility [10], s well s directly or indirectly decresing the contminnt iovilility in wter nd soil. Additionlly, iochr is renewle mteril tht is low cost, sustinle, nd environment friendly. It could e n efective wy of utiliztion of griculturl nd forestry wstes nd hs wide ppliction in sustinle griculture. Previous studies [1, 11] hve shown tht iochr improved plnt growth nd inhiited migrtion nd phytotoxicity of hevy metls. But, it is yet unknown on how to pply iochr in polluted wter nd how much the ccumultion of cdmium (Cd) could e reduced in mjor qutic crops such s Asin lotus (Nelumo nucifer Gertn.). In ddition, no dt is ville out the optimum mount of iochr pplied in polluted environment in order to produce sfe food nd medicine from qutic plnts. With incresing humn helth issues cused y hevy metl contmintion, strict stndrd on the mximum of hevy metl content hs een widely estlished in food nd medicinl products. The upper limit set y Europen Commission in Regultion No. 1881/2006 ws t 0.10 nd 0.20 mg/kg wet weight in stem vegetles nd fresh hers, respectively. The Chinese Ministry of Helth relesed GB (Limiting Chemicl Contminnts in Food) with Cd cutof vlue of 0.1 nd 0.2 mg/kg in root nd lefy vegetles, respectively. Cd is one of the top ive toxic hevy metls with strong chemicl ctivity, high toxicity, nd diiculty to e degrded. Its contmintion in mjor crops such s rice hs ecome severe prolem in Chin in recent yers [12, 13], nd excessive Cd content hs limited the export of crops nd Chinese medicines. Therefore, it is of gret scientiic vlue nd prcticl signiicnce to investigte food sfety nd pollution remedition in hevy metl-contminted environment. Asin lotus is widely grown in Chin, Jpn, nd other Asin countries s n importnt vegetle, medicinl, nd ornmentl plnt. Nerly, ll prts of lotus plnt re edile or used for medicinl purpose [14]. Lotus deserves tention ecuse of its economic potentil nd ecuse its product qulity nd economic vlue re inluenced y hevy metl pollution [15, 16]. Slvi miltiorrhiz Bunge (dnshen in Chin, red sge or Chinese sge in other res) hs een widely used s one of the most importnt trditionl Chinese hers in Chin, Kore, Jpn, nd other Asin countries [17]. Its dried roots contin mjor ctive constituents including vrious kinds of tnshinones tht phrmcologiclly function s tretment for ischemic crdiovsculr diseses, microcircultory disturnce-relted diseses, nd coronry disorders [17, 18]. Cd hs strong chemicl ctivity nd potentilly high toxicity tht cretes chllenges in remedition. Contmintion of Cd is one of the most serious issues ecuse it is mjor iohzrd in Chin. To investigte efects of iochr on the growth of Asin lotus nd Chinese sge, we conducted tril in rtiicilly Cd-polluted condition in continers to evlute the toxicity nd

4 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus ccumultion of Cd in oth qutic nd terrestril plnts. The results of this study my provide useful reference on restortion of hevy metl-polluted environment nd production of sfe nd orgnic griculturl products. 2. Mteril nd methods 2.1. Site detils The experiment ws conducted in plstic tunnel house nd greenhouse, respectively, which re locted t Shnghi Chenshn Plnt Science Reserch Center ( N; E), Chinese Acdemy of Sciences, nd Chenshn Botnicl Grden, Shnghi of Chin. The light intensity is 7000 lx ( lx) for oth tunnel house nd greenhouse during dylight hours. The temperture t 28 C (22 42 C) nd humidity t 50% (30 70%) re for tunnel house, nd the temperture t 25 C (20 36 C) nd humidity t 60% (40 80%) re for greenhouse throughout the experiment Plnt mteril N. nucifer Tikong Lin 36', one of the most importnt lotus cultivrs in Chin, nd Chinese sge (S. miltiorrhiz Bunge.), medicinl plnt, were used for experimentl plnts. The lotus seeds were otined from Gungchng White Lotus Development Bureu, Jingxi of Chin, nd those of S. miltiorrhiz were otined from Northwest Agriculture nd Forestry University, Shnxi, Chin Tril design nd tretments Pinewood iochr from locl mrket ws sieved to use pieces out cm in size (Tle 1). Nturl soil (lom) ws ir-dried (Tle 2) nd lended to crete uniform mix fter removing rocks, twigs, lef liters, etc. After recording dry weight, the soil ws moistened to 40% mximum ield wter cpcity with deionized wter. The iochr ws then dded nd thoroughly mixed with the soil t difering mounts: 0, 4, 8, 16, nd 32% iochr-soil (v/v), nd leled s CK (control), B0, B4, B8, B16, nd B32, respectively. The lotus seeds germinted in smll continers, nd the seedlings with three to four loting leves were trnsplnted in lrger pots without dringe holes for experimentl tretment in erly June, one plnt per pot nd six repliction. Forty dys lter, except CK, ech pot ws dded y the solution of 1.09 mg Cd(NO 3 ) 2 4H 2 O (3 mg/kg Cd). The pots were plced under trnsprent plstic tunnel house to void rinfll nd regulrly wtered y tp wter to retin the strting wter level. No fertilizer ws pplied throughout tril. For tril on Chinese sge, the soil-iochr mixture ws mde sme s tht in lotus experiment. One month lter, the dissolved Cd(NO 3 ) 2 4H 2 O (3 mg/kg Cd) ws dded to soil-iochr mixture other thn CK. The lotus seedlings with three to four leves were trnsplnted to the experimentl pots with sucers in June, one seedling ech pot nd six pots per tretment. Pots were rndomly rrnged. Throughout experiment, plnts were not fertilized ut wtered regulrly with tp wter to mintin soil moisture. To

5 52 Engineering Applictions of Biochr void possile leching of hevy metl nd other nutrients, the lechte from the pots ws collected nd lter repplied to the pots. ph Orgnic mter (cmol/kg) Ction exchnge cpcity (cmol/kg) Electricl conductivity Pore structure (ms/cm) (%) Cd content (mg/kg) 9.2 Immesurle Lrge 39%, smll 44% 0.17 Tle 1. Biochr properties. Totl nitrogen (g/kg) Totl phosphorus (g/kg) Totl potssium (g/kg) ph Orgnic mter (cmol/kg) CEC (cmol/kg) Electricl conductivity (ms/cm) Cd content (mg/kg) Tle 2. Soil properties Dt collection Plnt growth indictors In lotus experiment, lef numer, lef re, nd plnt height (equl to the height of the tllest lef) were recorded t 35, 75, nd 115 d, respectively, fter trnsplnting. Lef dimeter nd plnt height were mesured y ruler. Six mture emergent leves were rndomly selected from ech of the three pots to record lef dimeter ([long + short length dimeter]/2), nd then lef re (πr 2 ) ws clculted. Plnt height ws mesured from the top of the pot to the top of the highest lef in ech pot. Seventy dys fter dding Cd, ll the oveground prts nd underground prts (rhizomes only) were seprtely hrvested. After soil removl, the rhizomes were immersed in 0.2 μm EDTA solution for 30 min, followed y ir-drying for 12 h, nd then the fresh mss ws determined. Therefter, the weighed rhizomes were immeditely plced into envelopes for drying t 65 C for 84 h in n oven (DHG-9240A, Shnghi Yiheng Scientiic Instruments Co., Ltd., Chin), nd then the dry mss ws recorded. In Chinese sge experiment, lef numer nd re were recorded t 10, 35, nd 60 d, respectively, fter trnsplnting. The lef re of oth the lrgest nd smllest mture leves in ech pot ws mesured y lef re meter (YMJ-A, Zhejing Top Instrument Co., Ltd) to get the verge vlue for ech tretment (six plnts). Eighty dys fter trnsplnting, leves with stems nd roots were hrvested seprtely. After soil removl, root fresh weight ws determined efore immersing roots in 0.2 μm EDTA solution for 30 min followed y ir-drying for 4 h. Roots were then plced into envelopes nd dried t 65 C for 48 h in drying oven. Finlly, the root dry weight ws mesured Plnt physiologicl indictors All plnt smples were collected with hole puncher (1 cm dimeter) t the sme position of ech fresh lef. Wter extrct of leves ws used to mesure cell memrne permeility

6 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus y tissue luid exosmosis conductnce method (n = 3/tretment) using conductivity meter (DDS-11AT, Shnghi, Chin) [19]. The reltive chlorophyll content of fresh leves ws recorded y hndheld chlorophyll meter SPAD-502 Plus (Konic Minolt Sensing, Inc., Jpn; n = 6), ccording to Xiong [19]. Following Xiong [19], the thiorituric cid method (n = 3) ws used to determine methne dicroxylic ldehyde (MDA) content in fresh leves nd the NBT photochemicl reduction method to nlyze superoxide dismutse (SOD) ctivity. At the sme time s the chromogenic rection, enzyme-extrcted lef liquid ws collected nd tested t 560 nm OD y n ELISA plte reder (Tecn M200 PRO) (n = 3) [19] Cd content mesurements Cd contents in the rhizomes, roots, leves, nd petioles were determined y Grphite Furnce Atomic Asorption Spectrometry, ccording to the Determintion of Cdmium in Foods GB/T [20]. Microwve digestion method ws crried out with PE Anlyst AA800 (Perkin Elmer) (n = 3/tretment). The ioconcentrtion fctor (BCF) relects the degree or cpility of ccumultion of hevy metls from soil or wter y plnts. The iologicl trnsfer fctor (TF) refers to the trnsference of hevy metls in plnts in vivo Zyed et l. [21]. BCF nd TF were clculted y BCF = trce element concentrtion in plnt tissues t hrvest/initil concentrtion of the element in the externl nutrient solution nd TF = hevy metl content rtio (Cd in oveground tissues/cd in rhizomes or roots) Dt nlysis The verges were determined y Microsoft Excel The nlysis of vrince (ANOVA), Duncn s multiple rnge test, nd regression nlysis were conducted y sttisticl pckge for socil scientists (SPSS 16.0). Sttisticl signiicnce ws set t level of α = Results 3.1. Biochr promoted the growth of Asin lotus nd Chinese sge Lef growth In lotus experiment, no pprent lef toxicity ws oserved 5 dys fter dding Cd. Thirty-ive dys lter, the numer, height, dimeter, nd lef re of emerging leves of N. nucifer Tikong Lin 36 reched the highest in the tretments with 32% iochr (Tle 3). But, the reltionship ws uncler etween the numer of loting leves nd iochr rtio. Forty dys lter, the sme plnt indexes in tretment H0 were the lowest, only 32, 39, 59, nd 29% of tht in tretment H32, respectively. However, diferences were not signiicnt etween H0 nd the control. Therefore, Cd of low concentrtion could not cuse strong toxicity. The reltionships etween iochr nd the numer, height, dimeter, nd lef re of emerging leves in lotus could e relected y (1) y = 0.03 x x (R 2 = 0.955, P = 0.037), (2) y = x x (R 2 = 0.830, P = 0.170), (3) y = x x (R 2 = 0.836, P = 0.164), nd (4) y = x x (R 2 = 0.962, P = 0.037), respectively. It indicted tht the growth of lotus ws

7 54 Biochr rte (%) Floting lef numer Emerging lef numer Emerging lef height (cm) Lef dimeter (cm) Lef re (m 2 ) 35 d with iochr CK 8.2 ± 4.4 H ± 2.1 H4 7.7 ± d with iochr nd Cd 9.5 ± ± ± d with iochr nd Cd 11.5 ± ± ± d with iochr 6.3 ± ± ± d with iochr nd Cd 7.5 ± ± ± d with iochr nd Cd 11.7 ± ± ± d with iochr 8.4 ± 2.5 d 8.5 ± 3.6 d 16.7 ± 4.3 c 75 d with iochr nd Cd 9.0 ± 5.0 c 9.0 ± 6.6 c 21.2 ± 7.6 c 115 d with iochr nd Cd 21.8 ± 10.7 c 22.3 ± 10.4 c 39.5 ± d with iochr 6.7 ± 0.7 d 7.0 ± 1.1 d 7.9 ± 0.9 cd 75 d with iochr nd Cd 9.2 ± 1.4 c 9.4 ± 0.8 c 11.3 ± 1.4 c 115 d with iochr nd Cd 11.0 ± 1.4 c 12.1 ± 2.5 c 15.4 ± d with iochr 0.1 ± 0.0 c 0.1 ± 0.0 c 0.1 ± 0.0 c 75 d with iochr nd Cd 0.1 ± 0.1 c 0.1 ± 0.1 c 0.2 ± 0.1 c 115 d with iochr nd Cd 0.2 ± 0.1 d 0.4 ± 0.5 c 0.9 ± 0.4 Engineering Applictions of Biochr H ± ± ± ± ± ± ± 6.6 cd 17.0 ± 8.7 c 33.0 ± 18.2 c 8.4 ± 0.8 c 11.5 ± 2.7 c 15.1 ± ± 0.1 c 0.3 ± 0.1 c 0.7 ± 0.3 H ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.5 H ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.6 CK refers to none of iochr nd Cd dded to the soil; H0, H4, H8, H16, nd H32 refer to dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters in the sme column indicte signiicnt diference (P < 0.05) mong tretments. Tle 3. Efects of iochr on lef growth of N. nucifer Tikong Lin 36 in Cd-polluted wter.

8 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus signiicntly promoted y iochr dded t 16 32% (with Cd dded). The higher rtios of iochr promoted more lef growth ut lso incresed the cost (iochr cost). In the experiment on Chinese sge, morphologicl lef toxicity did not occur 10 d fter dding Cd nd iochr tretments. Both lef numer nd re incresed t 35 d fter tretment nd reched the highest vlue in the tretments with the highest iochr proportion (B32) t 60 d for the inl mesurement (Tle 4). At 60 d, the lef numer of B4, B8, B16, nd B32 incresed 26.2, 30.9, 37.8, nd 42.9%, respectively, while tht of CK nd B0 incresed 19.4 nd 26.0%, respectively, compred with those t 10 d. Likewise, lef re of B4, B8, B16, nd B32 incresed 36.3, 50.9, 70.7, nd 69.9%, respectively, while tht of CK nd B0 just incresed 28.3 nd 31.0%, respectively. Therefore, lef growth of S. miltiorrhiz ws signiicntly promoted with the ddition of 16 32% iochr (with Cd dded). Lower concentrtions of Cd did not cuse strong toxicity prolems ut resulted in the smller lef size nd slower growth of plnts Plnt iomss Biomss discrepncy is the most direct response to vrious environmentl stimuli for plnts. In lotus, our study showed tht the totl fresh weight, the fresh weight of the oveground prts, nd the fresh nd dry weight of rhizomes incresed y dding iochr (Figure 1). All the highest vlues of the tested plnt growth indexes occurred in tretment H32. Among which, the highest totl fresh weight ws 4.5 times greter thn H0. The fresh weight of underground prts mde the gretest contriution to the totl fresh weight, which correlted signiicntly with the proportion of iochr, nd the mximum ws 6.25 times greter thn control vlues. In Chinese sge, the totl fresh weight, the lef fresh weight, nd the fresh nd dry weight of roots incresed with the ddition of iochr in our study (Figure 2). All the highest vlues of the tested plnt growth indices occurred in tretment B32 where the highest totl fresh weight ws 1.3 times of tht in tretment B0. The fresh weight of roots mde greter contriution to Biochr rte/% Lef numer Lef re (cm 2 ) 10 d 35 d 60 d 10 d 35 d 60 d CK 19.3 ± ± 3.2 c 24.3 ± ± ± ± 5.6 c B ± ± ± ± ± 3.7 c 41.3 ± 4.0 c B ± ± 2.9 c 26.5 ± ± ± 5.2 c 43.9 ± 6.4 c B ± ± ± ± ± 7.2 c 51.2 ± 4.5 B ± ± ± ± ± 4.2 c 60.0 ± 3.8 B ± ± ± ± ± ± 4.1 CK indictes no iochr nd no Cd in the soil; B0, B4, B8, B16, nd B32 indicte dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters in the sme column indicte signiicnt diferences (P < 0.05) mong tretments. Tle 4. Efects of iochr on lef growth of S. miltiorrhiz in Cd-polluted soil ( x ± s).

9 56 Engineering Applictions of Biochr Weight (g) Totl fresh weight Lef fresh weight Rhizome fresh weight Rhizome dry weight c c c c d d c d c d c c c CK H0 H4 H8 H16 H32 Figure 1. Efects of iochr on plnt weight of N. nucifer Tikong Lin 36 in Cd-polluted wter. CK refers to none of iochr nd Cd dded to the soil; H0, H4, H8, H16, nd H32 refer to dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters indicte signiicnt diference (P < 0.05) mong tretments Totl fresh mss Lef fresh mss Root fresh mss Root dry mss Weight (g) CK B0 B4 B8 B16 B32 Figure 2. Efects of iochr on mss of S. miltiorrhiz in Cd-polluted soil. CK indictes no iochr nd no Cd in soil; B0, B4, B8, B16, nd B32 indicte dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters indicte signiicnt diferences (P < 0.05) mong tretments. the totl fresh weight thn tht of the leves nd stems, nd it correlted signiicntly with the iochr proportion. The mximum root fresh nd dry weight oserved in B32 ws 1.4 times of tht in the control, which further veriied tht iochr promoted root growth.

10 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus Efects of iochr on physiologicl sttus Chlorophyll content The reltive content of chlorophyll cn e used to evlute plnt growth, physiologicl chnges, nd nitrogen nutrition. Three consecutive mesurements on lef chlorophyll were tken y SPAD t 20, 50, nd 80 d fter dding iochr (Tle 5). The reltive chlorophyll contents in leves of treted plnts were signiicntly higher thn those in the control, ut no signiicnt difference ws found etween iochr tretments. The highest vlues in ech tretment occurred t 10 d fter dding Cd when the lef growth reched its pek. The highest chlorophyll content (82%) in H0, followed y 62% in H16, could e explined y tht Cd did not rech toxic stress concentrtion fecting the enzyme ctivity relevnt to chlorophyll formtion. On the contrry, low concentrtion of Cd promoted chlorophyll synthesis. Biochr incresed the reltive chlorophyll content of lotus lef. The chlorophyll formtion ws signiicntly promoted t 20 nd 50 d fter iochr eing dded. Fifty dys lter, it decresed slowly s plnts strted senescence. In Chinese sge, the reltive chlorophyll content ws tested t 10, 35, nd 60 d, respectively, fter trnsplnting (Tle 6). Reltive chlorophyll contents in the leves of treted plnts were higher thn those in the control, ut no diference ws found etween the tretments of iochr rtios. The highest chlorophyll content in ll tretments occurred t 35 d when the leves were t the pek stge of growth. The highest increse rte of chlorophyll content from 10 to 35 d ws in tretment B32 (42%), indicting tht Cd might fect the enzyme ctivity relevnt to chlorophyll synthesis, nd it stimulted the chlorophyll formtion t certin concentrtion. Sixty dys fter iochr tretment, the reltive chlorophyll content egn to decrese slowly s the plnts grdully senesced Cell memrne permeility In oth lotus nd Chinese sge, the mximum of cell memrne conductivity occurred in tretment H0 nd ws signiicntly diferent from the control nd iochr tretments (Figure 3A nd B). This Biochr rte (%) Reltive chlorophyll content (20 d with iochr) Reltive chlorophyll content (50 d with iochr) Reltive chlorophyll content (80 d with iochr) CK 12.7 ± ± ± 0.5c H ± ± ± 1.4c H ± ± ± 0.7 H ± ± ± 0.8 c H ± ± ± 0.9 H ± ± ± 0.27 c CK refers to none of iochr nd Cd dded to the soil; H0, H4, H8, H16, nd H32 refer to dding 0, 4, 8, 16 nd 32% of iochr in soil, respectively. Diferent lower-cse leters in the sme column indicte signiicnt diference (P < 0.05) mong tretments. Tle 5. Efects of iochr on reltive chlorophyll content of N. nucifer Tikong Lin 36 in Cd-polluted wter.

11 58 Engineering Applictions of Biochr Biochr rte (%) Reltive chlorophyll content 10 d 35 d 60 d CK 16.9 ± ± ± 0.4 B ± ± ± 0.5 B ± ± ± 0.9 c B ± ± ± 0.9 c B ± ± ± 0.9 c B ± ± ± 0.4 CK indictes no iochr nd no Cd in soil; B0, B4, B8, B16, nd B32 indicte dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively; nd diferent lower-cse leters in the sme column indicte signiicnt diferences (P < 0.05) mong tretments. Tle 6. Efects of iochr on reltive chlorophyll content of S. miltiorrhiz in Cd-polluted soil. demonstrted tht 3 mg/kg of Cd stressed the plnts resulting in n increse of memrne permeility. Our dt showed tht dding iochr could reduce cell memrne conductivity, ut no chnge trend is relted to the iochr rtio MDA content Figure 4 showed the efects of diferent proportions of iochr on MDA content of N. nucifer Tikong Lin 36 grown for 20 d in Cd-polluted wter nd S. miltiorrhiz grown for 30 d in Cd-polluted soil. The highest MDA content ws seen in the tretment H8 nd the lowest in H32 for lotus (Figure 4A). However, no signiicnt diferences in MDA content were found etween tretments where oth Cd nd iochr were dded, which indicted tht the mount of Cd did not produce gret impct on the cell memrne lipid peroxidtion. The other possile reson ws tht the physiologicl metolic ctivity of N. nucifer Tikong Lin 36 ws distured y Cd, leding to the ccumultion of oxidtion product MDA, while the MDA ccumultion ws inhiited y iochr, which llevited the dmge of the memrne system cused y lipid memrne peroxidtion. In Chinese sge, the highest MDA content ws seen in CK (Figure 4B), which indicted tht the mount of Cd did produce some impct on the lipid peroxidtion of cell memrnes. Less MDA content occurred in iochr tretments, nd the lowest ws seen in B16, lthough no diferences were found mong the tretments with oth Cd nd iochr. The possile reson for no difference in tretments might e tht the physiologicl metolic ctivity ws distured y the MDA ccumultion due to Cd ddition, while the MDA ccumultion ws inhiited y iochr, which llevited the dmge of the memrne system cused y lipid memrne peroxidtion SOD ctivity When oth N. nucifer Tikong Lin 36 nd S. miltiorrhiz grew for 30 d in Cd-contminted environment, the efects of iochr on SOD ctivity in fresh leves reched signiicnt level

12 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus A Lef cell memrne permeility (us/cm) CK H0 H4 H8 H16 H32 Lef cell memrne permeility (us.cm -1 ) B CK B0 B4 B8 B16 B32 Figure 3. Efects of iochr on lef cell memrne permeility of N. nucifer Tikong Lin 36 in Cd-polluted wter (A) nd S. miltiorrhiz in Cd-polluted soil (B). CK refers to none of iochr nd Cd dded to the soil; H0, H4, H8, H16, nd H32 refer to dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters indicte signiicnt diference (P < 0.05) mong tretments. (Figure 5). The highest SOD ctivity ppered in tretment H0, which indicted tht Cd signiicntly enhnced the SOD ctivity of plnts. However, fter dding iochr, SOD ctivity declined signiicntly, with the highest decrese y 53% in tretment H8 for lotus (Figure 5A) nd y 29% in tretment B16 for Chinese sge (Figure 5B), compring to the control H0, though decrese trend ws inconsistent with the proportion of iochr Cd content The Cd ccumultion in plnts clerly incresed fter dding Cd ut signiicntly decresed when iochr is dded (Figures 6 nd 7). For N. nucifer Tikong Lin 36', the Cd content in rhizomes, petioles, nd leves incresed y 12.81, 17.35, nd 7.56 folds, respectively, in tretment H0, compred with the control (Figure 6). With incresing iochr, Cd in plnt tissue decresed correspondingly. In H32 tretment, the Cd content in rhizomes ws mg/kg,

13 60 Engineering Applictions of Biochr MDA content (umolg fw -1 ) A 0 CK H0 H4 H8 H16 H B MDA content (umolg fw -1 ) CK B0 B4 B8 B16 B32 Figure 4. Efects of iochr on MDA content in lef of N. nucifer Tikong Lin 36 in Cd-polluted wter (A) nd S. miltiorrhiz in Cd-polluted soil (B). CK refers to none of iochr nd Cd dded to the soil; H0, H4, H8, H16, nd H32 refer to dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters indicte signiicnt diference (P < 0.05) mong tretments. only 68.51% of tht in H0. It ws more reduced in petioles from to mg/kg, decrese of 81.41%. The lowest Cd content in leves ws mg/kg in H32 nd decresed y 54.66% of the highest in H0. However, the Cd content in ll evluted plnt prts remined ove the limit set y the Europen Commission regultions. The reltionship for the proportion of iochr (x) reltive to Cd content (y) in rhizomes could e relected y (5) y = x x x (R 2 = 0.999, P = 0.351). Bsed on this reltionship, higher level of iochr possily further reduces Cd uptke nd mkes the

14 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus A SOD ctivity (U g -1 ) c c c 0.2 d CK H0 H4 H8 H16 H32 SOD content (U g -1 ) B c c CK B0 B4 B8 B16 B32 Figure 5. Efects of iochr on SOD ctivity in the lef of N. nucifer Tikong Lin 36 in Cd-polluted wter (A) nd S. miltiorrhiz in Cd-polluted soil (B). CK refers to none of iochr nd Cd dded to the soil; H0, H4, H8, H16, nd H32 refer to dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters indicte signiicnt diference (P < 0.05) mong tretments. rhizomes of lotus grown in Cd contminted condition meet food sfe stndrds. The reltionship etween the proportion of iochr (x) nd Cd content (y) in petioles nd leves could e presented y (6) y = x x x (R 2 = 0.965, P = 0.238) nd (7) y = x x x (R 2 = 0.793, P = 0.559), respectively. In Chinese sge, Cd content in roots nd leves ws 0.57 nd 0.18 mg/kg, respectively, in tretment B0 nd incresed to 2.2 nd 2.0 folds compred with the control (Figure 7). However, with incresing iochr, Cd content in plnt tissues decresed correspondingly. In roots, Cd content ws the lowest 0.25 mg/kg in B32, only 43.6% of tht in B0. It ws much lower in leves

15 62 Engineering Applictions of Biochr Cd content (mg kg-1) d e c c c Cd content in the rhizome Cd content in the petiole Cd content in the lef CK H0 H4 H8 H16 H32 c d c d Figure 6. Efects of iochr on Cd content in the root, petiole, nd lef of N. nucifer Tikong Lin 36 in Cd-polluted wter. CK refers to none of iochr nd Cd dded to the soil; H0, H4, H8, H16, nd H32 refer to dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters indicte signiicnt diference (P < 0.05) mong tretments Cd content in the root Cd content in the lef Cd content (mg kg -1 ) d c cd d 0.1 d c c d 0.0 CK B0 B4 B8 B16 B32 Figure 7. Efects of iochr on Cd content in the root nd lef of S. miltiorrhiz in Cd-polluted soil. CK indictes no iochr nd no Cd in soil; B0, B4, B8, B16, nd B32 indicte dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters indicte signiicnt diferences (P < 0.05) mong tretments. in the sme tretment, down to 0.09 mg/kg, decrese of 52.8% of the highest in B0, nd ws elow the upper limit vlue set y the Chinese Phrmcopoei [22]. The regression equtions for the proportion of iochr (x) relted to Cd content (y) in roots nd leves, respectively, were y = x x x ( R 2 = 0.997, P = ) (8) y = x x x ( R 2 = 0.991, P = ) (9)

16 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus The incresing proportion of iochr in 3 mg/kg Cd-contminted plnting mix likely further prevents Cd uptke to mke the roots ( mjor medicinl prt) meet sfe stndrds of Chinese medicine Cd BCF nd S/R The BCF shows the cpcity of plnts to dsor hevy metls. In lotus cse, the BCF ws the lowest in the rhizomes of the control, only 7.6% of the highest vlue of H0 (Figure 8). However, fter dding iochr, the BCF decresed signiicntly, with the lowest (0.042) in H32, out one-third of tht in the control. The highest BCF (0.385) of the ove-ground prts occurred in H0 nd ws 11 times greter thn tht in the control. Likewise, the ove-ground BCF ws reduced signiicntly, with the iggest decrese seen in H32, down to 70.65% of tht in the control. The petiole s BCF ws signiicntly less thn tht of the leves. The trnsfer coeicient from roots to shoots (S/R) showed tht Cd ws trnsported to diferent orgns of plnts. The S/R rose with the incresing iochr proportions nd the highest vlue (3.06) ws oserved in H32. The increse of S/R ws not signiicnt, ut it indicted tht iochr could fect Cd trnsference nd reduce its ccumultion in rhizomes. In Chinese sge, the highest BCF (0.19) of the roots occurred in B0, 2.2 times tht of the control (Figure 9), indicting tht roots were le to ccumulte Cd. However, fter dding iochr, the BCF of oth root nd lef decresed with the rte of iochr. The lowest root BCF (0.08) in tretment B32 ws only 43% of the highest oserved in B0. Likewise, the lowest BCF of lef BCF of the rhizome BCF of the ovegroud S/R Vlue CK H0 H4 H8 H16 H32 Figure 8. Efects of iochr on Cd ioconcentrtion fctor (BCF) nd iologicl trnsfer coeicient (S/R) of N. nucifer Tikong Lin 36 in Cd-polluted wter. CK refers to none of iochr nd Cd dded to the soil; H0, H4, H8, H16, nd H32 refer to dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. Diferent lower-cse leters indicte signiicnt diference (P < 0.05) mong tretments.

17 64 Engineering Applictions of Biochr BCF of the root BCF of the lef TF Vlue CK B0 B4 B8 B16 B32 Figure 9. Efects of iochr on Cd ioconcentrtion fctor (BCF) nd iologicl trnsfer fctor (TF) of S. miltiorrhiz in Cd-polluted soil. CK refers to no iochr nd no Cd in soil; B0, B4, B8, B16, nd B32 indicte dding 0, 4, 8, 16, nd 32% of iochr in soil, respectively. lso occurred in B32, 77.5% of the highest in B0. It is possily due to the cpcity of iochr to inhiit sorption of Cd in plnts. The trnsfer fctor (TF) from roots to leves showed tht Cd cn e sored from soil y S. miltiorrhiz nd trnsported mong diferent plnt orgns. The highest TF ws oserved in the control (0.65). TF decresed with the ddition of Cd. This could e explined y Cd potentilly inhiiting growth nd metolism of S. miltiorrhiz, therefore slowing the sored Cd from the roots. With the incresing iochr proportion, TF decresed correspondingly. The lowest TF ws 0.3 in B32, less thn hlf of tht in the control, indicting tht iochr could fect the trnsfer of Cd from roots to leves. 4. Discussion 4.1. Efects of iochr on plnt growth In this study, 3-mg/kg Cd dded to the soil or soil-wter mix did not elicit overt symptoms of hevy metl poisoning such s lef chlorosis or stunting in lotus ut resulted in treted plnts eing stunted nd undersized in Chinese sge when compred to CK nd iochr tretments. The lck of oserved toxicity in N. nucifer my e due to its strong resistnce of the polluted environments, such s in Cd-polluted cse study [23]. For oth cses of our study, the lef growth, totl fresh weight, oveground fresh weight, nd fresh nd dry weight of underground prts of plnts incresed with iochr rtio, nd nerly ll the highest vlues ppered in the tretment with the highest mount of iochr. Therefore, iochr cn

18 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus promote plnt growth, which hs lso een reported in the previous studies [7, 24]. Becuse no fertilizer ws pplied fter dding iochr, the iomss increse suggests tht iochr might work s fertilizer [25] or fertilizer enhncer [24], y meliorting soil ph, CEC, se sturtion, nd other physicochemicl properties, y fecting soil nutrient vilility, such s the efectiveness of K, C, N, Mg, N, P, nd other elements [26], or y incresing the rhizosphere nd soil nitrogen retention indirectly [27]. Yet some studies lso showed tht iochr hd no eneicil efect on plnt iomss, which is relted to the type of soil nd iochr, fertility levels, nd other relevnt experimentl conditions [5, 28, 29]. Although Cd inhiited the underground growth of lotus, dding iochr ofsets the stress of Cd nd signiicntly promoted the rhizome enlrgement nd iomss increse. The 16 32% of iochr dded in soil ws more eneicil for growth of oth lotus nd Chinese sge. However, more iochr ddition will increse production cost Efects of iochr on physiologicl sttus Cell memrne permeility, reltive chlorophyll content, MDA, nd SOD served s mrkers to represent plnt rections under stress conditions. Our study showed tht Cd dded to wter or soil produced stress on cell memrne structure of plnt, ut no signiicnt diference occurred mong tretments with diferent proportions of iochr. Therefore, no optiml iochr level could e determined sed on this point. Possily, the eneicil efects of iochr on memrne permeility were lso fected y other environmentl fctors, such s ph, EC, etc., mking the memrne permeility chnge irregulrly. The chlorophyll content in plnt leves incresed with iochr mount in ll tretments, which coincided with the previous report, in which low Cd concentrtion fcilitted chlorophyll formtion nd high Cd concentrtion reduced chlorophyll content [23, 30]. This phenomenon lso occurred in other hevy metl study cses [31]. In the erly period of plnt growth, promotion efect of iochr on chlorophyll ws not evident. However, lef chlorophyll content incresed in the middle period, followed y reltively ig decrese in the lter. Therefore, iochr promoted plnt growth nd dvnced plnt senescence simultneously. In lotus cse, fter dding Cd nd iochr, the MDA contents showed no diference mong tretments ecuse low Cd concentrtions did not increse cell memrne peroxidtion. Therefore, mitigting efect of iochr could not e determined. But, in the cse of Chinese sge, the signiicnt diferences showed in MDA content etween tretments with nd without iochr, which suggested tht iochr llevited cell memrne peroxidtion cused y Cd. In oth cses, SOD ctivity signiicntly incresed y dding Cd, indicting tht Cd stimulted the physiologicl defense mechnisms nd physiologicl resistnce ctivities nd improved ctivity of ntioxidnt nd its enzyme [30, 32]. However, the ddition of iochr signiicntly decresed SOD ctivity, which suggested tht iochr cn meliorte Cd hzrds Efects of iochr on Cd content in plnts Cd ccumultion ws oserved in the orgns nd tissues of N. nucifer Tikong Lin 36 nd S. miltiorrhiz in rtiicilly polluted environment, nd the ccumultive concentrtions incresed more thn 10 folds in some tretments when compred with the control. The uptke of Cd ws

19 66 Engineering Applictions of Biochr signiicntly reduced y dding iochr in ll tretments, ut the mximum efect ws oserved in H32. The Cd content in roots of S. miltiorrhiz could e reduced to meet the mximum llowed vlue set y the Chinese Phrmcopoei [22]. In oth cses, Cd content in plnts could possily e further decresed with more iochr dded, extrpolting from the regression eqution of iochr nd Cd content. The role of iochr on reducing the hevy metl ccumultion in plnts hs een demonstrted in the previous reports [33, 34]. It might e relted to huge speciic surfce re nd strong dsorption functions of iochr to form insolule cheltes or cuse Cd precipittion. Biochr reduced the iovilility of Cd in soil or wter, y fecting ph, CEC (ction exchnge cpcity), wter-holding cpcity, nd other physicochemicl properties of the soil. Although the Cd-contminted lotus rhizomes in our study did not meet the edile stndrd, the regression eqution of iochr nd Cd content indicted tht it ws possile to further reduce the Cd content to the sfe rnge if more iochr would e pplied or the environment is less polluted. Comined with Cd contents in diferent orgns of N. nucifer Tikong Lin 36, ll the lowest BCFs in vrious orgns occurred in the controls, while the highest in tretments H0. In Chinese sge, BCFs of the root nd lef decresed with the ddition of iochr indicte tht esides its strong ility of dsoring Cd, iochr lso reduced the iovilility of hevy metls or immoilized hevy metls y chnging the physicochemicl property of soil [35]. The highest S/R vlues were oserved in the tretment with 32% iochr (H32), nd the lowest showed in the tretment H0. Bsed on the vlues of Cd content, Cd BCF, nd S/R, 32% of iochr ws the optimum mount for pplying to demonstrte iochr s potentil in plnt growth promotion, ventiltion nd wter retention, fertilizer eiciency, nd pollution reduction. 5. Conclusion In this study, the efects of iochr were evluted on edile nd medicinl plnts. The mjor gols were to understnd growth-promoting efects of iochr nd investigte how iochr llevites the toxicity nd ccumultion of Cd in rtiicilly Cd-mended pot culture. The results showed tht the contents of Cd in rhizomes, petioles, nd leves of N. nucifer Tikong Lin 36 nd in roots, leves, nd stems of S. miltiorrhiz were signiicntly reduced nd the trnsference of Cd in plnts ws inhiited y dding iochr. Although the impcts of iochr on plnt physiologicl indictors were not very cler, iochr not only promoted plnt growth ut lso llevited toxic efects of Cd in Cd-contminted environment. Therefore, s vlule soil mendment, iochr hs huge potentil in green griculture production nd remedition of hevy metl-polluted wter or soil due to its positive efects of improving plnt growth nd reducing hevy metl uptke. Further studies re needed to evlute efects of iochr on sfe production of lotus in the ield nd other importnt vegetles, fruit trees, nd Chinese medicinl plnts nd to estlish the optimum of physiologiclly nd economiclly fesile mount of iochr to dd for environmentl remedition. Acknowledgements This study ws funded y Shnghi Administrtion Bureu of Lndscpe nd City Appernce.

20 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus Author detils Dike Tin 1,2 *, Amei Liu 1,2 nd Ynci Xing 3 *Address ll correspondence to: dktin@sis.cn.cn 1 Shnghi Chenshn Plnt Science Reserch Center, Chinese Acdemy of Sciences, Shnghi, Chin 2 Shnghi Key Lortory for Plnt Functionl Genomics nd Resources, Shnghi Chenshn Botnicl Grden, Shnghi, Chin 3 School of Life Science, Hunn University of Science nd Technology, Xingtn, Hunn, Chin References [1] Beesley L, Moreno-Jiménez E, Gomez-Eyles JL. Efects of iochr nd green wste compost mendments on moility, iovilility nd toxicity of inorgnic nd orgnic contminnts in multi-element polluted soil. Environmentl Pollution. 2010; 158(6): [2] Lohmnn R, Mcfrlne JK, Gschwend PM. Importnce of lck cron to sorption of ntive PAHs, PCBs, nd PCDDs in Boston nd New York hror sediments. Environmentl Science nd Technology. 2005;39: [3] Lu H, Li Z, Fu S, Méndez A, Gscó G, Pz-Ferreiro J. Efect of iochr in cdmium vilility nd soil iologicl ctivity in n nthrosol following cid rin deposition nd ging. Wter Air Soil Pollution. 2015;226: [4] Mohn D, Pitmn Jr. CU, Brick M, Smith F, Yncey B, Mohmmd J, Steele PH, Alexndre-Frnco MF, Gómez-Serrno V, Gong H. Sorption of rsenic, cdmium, nd led y chrs produced from fst pyrolysis of wood nd rk during io-oil production. Journl of Colloid nd Interfce Science. 2007;310:57-73 [5] Alurquerque JA, Slzr P, Brrón V, Torrent J, del Cmpillo MC, Gllrdo A, Villr R. Enhnced whet yield y iochr ddition under diferent minerl fertiliztion levels. Journl of Plnt Nutrition nd Soil Science. 2013;177:16-25 [6] Ventur F, Slvtorelli F, Pin S, Pieri L, Pis PR. The efects of iochr on the physicl properties of re soil. Erth nd Environmentl Science Trnsctions of the Royl Society of Edinurgh. 2012;103(01):5-11 [7] Hung M, Yng L, Qin HD, Jing LG, Zou YB. Fertilizer nitrogen uptke y rice incresed y iochr ppliction. Biology nd Fertility of Soils. 2014;50(6): [8] Steineiss S, Gleixner G, Antonieti M. Efect of iochr mendment on soil cron lnce nd soil microil ctivity. Soil Biology & Biochemistry. 2009;41(6):

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22 Effects of Biochr on Plnt Growth nd Cdmium Uptke: Cse Studies on Asin Lotus [24] Utomo Widowti WH, Guritno B, Soehoo LA. The efect of iochr on the growth nd N fertilizer requirement of mize (Ze mys L.) in green house experiment. Journl of Agriculturl Science. 2012;4(5): [25] Lehmnn J. Bio-energy in the lck. Frontiers in Ecology nd the Environment. 2007;5(7): [26] Oguntunde PG, Fosu M, Ajyi AE, Gesen N. Efects of chrcol production on mize yield, chemicl properties nd texture of soil. Biology nd Fertility of Soils. 2004; 39(4): [27] Prendergst-Miller MT, Duvll M, Sohil SP. Biochr-root interctions re medited y iochr nutrient content nd impcts on soil nutrient vilility. Europen Journl of Soil Science. 2014;65(1): [28] Devereux RC, Sturrock CJ, Mooney SJ. The efects of iochr on soil physicl properties nd winter whet growth. Erth nd Environmentl Science Trnsctions of the Royl Society of Edinurgh. 2012;103(01):13-18 [29] Eyles A, Bound SA, Oliver G, Corkrey R, Hrdie M, Green S, Close DC. Impct of iochr mendment on the growth, physiology nd fruit of young commercil pple orchrd. Trees. 2015;29(6): [30] Yng SC, Wu Q. Efect of Cd on growth nd physiologicl chrcteristics of Aegicers conrnicultum seedlings. Mrine Environmentl Science. 2003;22(1):38-42 (in Chinese with English strct) [31] Jonker MT, Koelmns AA. Sorption of polycyclic romtic hydrocrons nd polychlorinted iphenyls to soot nd soot-like mterils in the queous environment: Mechnistic considertions. Environmentl Science nd Technology. 2002;36(17): [32] Li ZZ, Wu J, Tng Y, Yng G. Efect of P, Zn nd their interctions on the chlorophyll content nd ntioxidnt enzyme systems of Houtuyni cordt Thun. Act Ecologic Sinic. 2007;27(12): [33] Fellet G, Mrchiol L, Delle Vedove G, Peressoti A. Appliction of iochr on mine tilings: Efects nd perspectives for lnd reclmtion. Chemosphere. 2011;83(9): [34] Pug AP, Areu CA, Melo LCA, Pz-Ferreiro J, Beesley L. Cdmium, led, nd zinc moility nd plnt uptke in mine soil mended with sugrcne strw iochr. Environmentl Science nd Pollution Reserch. 2015;22(22): [35] Rees F, Simonnot MO, Morel JL. Short-term efects of iochr on soil hevy metl moility re controlled y intr-prticle difusion nd soil ph increse. Europen Journl of Soil Science. 2014;65(1):

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