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1 Agriculture nd Nturl Resources 51 (17) 7e5 Contents lists ville t ScienceDirect Agriculture nd Nturl Resources journl homepge: Originl Article Phytotoxic effects of rgn shell iochr on sld nd rley germintion Lil Bouqis,, * Slm Doud, Hns Werner Koyro, c Cludi Irene Kmmnn, c Ftim Zohr Ainlhout, Mouly Cherif Hrrouni d Polydisciplinry Fculty, In Zohr University, Troudnt, Morocco Fculty of Sciences, In Zohr University, Agdir, Morocco c Deprtment of Plnt Ecology, Justus-Lieig-University, Giessen, Germny d Hssn II Agronomic nd Veterinry Institute, Agdir, Morocco rticle info strct Article history: Received Septemer 1 Accepted 1 April 17 Aville online 15 My 17 Keywords: Argn shells Brley germintion Biochr Phytotoxic tests Sld germintion Biochr produced from rgn shells cn e contminted y toxic sustnces ccumulted during the pyrolysis process. To determine the potentil impct of toxic sustnces nd slt stress, this study focused on the effect rgn shell iochr hd on the germintion of sld (%,.5%, 1%, %, % or 8% iochr dry weight in snd-iochr mixture) nd rley seeds (%, 1%,.5%, 5% or 1% iochr dry weight in pet-iochr mixture). No negtive slt stress effect of rgn iochr on the germintion of sld ws oserved nor on the germintion rte nd fresh weight of seedlings. Additionlly, iochr ppliction incresed the germintion rte nd the fresh iomss weight in ll of the tretments. No significnt difference ws oserved from the control with the rley germintion rte, fresh nd dry weights of rley seedlings, wter content nd wter use efficiency of different mixtures (pet-iochr). Thus, for oth the sld nd rley germintion tests, no negtive effects of iochr produced from rgn shells were identified, providing preliminry indiction tht it could e sfely used for griculture. Copyright 17, Ksetsrt University. Production nd hosting y Elsevier B.V. This is n open ccess rticle under the CC BY-NC-ND license ( Introduction The ppliction of iochr to soils is ecoming more nd more common nd it is now generlly ccepted s mngement tool for the sequestrtion of cron nd the mitigtion of glol climte chnge in soils where the cron would otherwise hve een relesed into the tmosphere rpidly s cron emissions (Lorenz nd Ll, 1; Smith, 1). It hs lso een used to mend nutrient-poor soil for soil ecologicl restortion, soil ph, nutrient moility nd wter retention (Lehmnn et l., ; Liu et l., 1; Wng et l., 1). It cn lower chemicl fertilizer usge nd prevent eutrophiction of groundwter (Xu et l., 13; Shreef nd Zho, 17). Some studies hve shown tht it could lso increse the growth of plnts nd microil ctivity (Cui et l., 13; Zhng et l., 1), decrese soil ulk density (Brewer et l., 1; Liu et l., 1), increse ir-filled porosity (Hn et l., 15; Oi et l., 1) nd increse wter holding cpcity (Ael et l., 13; Wng et l., 1). Thus iochr improves the physicl, chemicl nd iologicl * Corresponding uthor. E-mil ddress: ouqisl@yhoo.fr (L. Bouqis). properties of soils, which hve n indirect effect on incresed yields (Smider nd Singh, 1; Zhng et l., 1). Under semi-rid, rid nd desert climtic conditions, such s in Morocco, griculturl prctices reserve mjority of the wter resources for irrigtion, with the resultnt extent of irrigted griculture comining with high evportive rtes leding to degrdtion of soil qulity (Bdroui et l., ). One possile solution is the ddition of iochr, highly porous pyrolyzed iomss which is well documented to help retin wter nd nutrients in soils. In previous reserch (Bouqis et l., 1), rgn shells otined fter extrction of rgn oil were selected s the iomss source nd converted into iochr using pyrolytic stove fricted in Morocco sed on the design provided y Dr. Cludi Kmmnn (Institute for Plnt Ecology, Giessen University, Germny). The physicl nd chemicl nlysis of rgn shell iochr reveled highly lkline ph (ph ¼ 1.7), high electricl conductivity (EC ¼.83 ms/cm), high contents of K (19.5 prts per million; ppm), N (339. ppm), Mg (15.89 ppm) nd NNO 3 (1 ppm), nd low contents of C (.8 ppm), KH PO (.33 ppm) nd of hevy metls compred to sndy soil nd pet. Thus, due to its high X/Copyright 17, Ksetsrt University. Production nd hosting y Elsevier B.V. This is n open ccess rticle under the CC BY-NC-ND license ( cretivecommons.org/licenses/y-nc-nd/./).

2 8 L. Bouqis et l. / Agriculture nd Nturl Resources 51 (17) 7e5 nutrient content nd low content of hevy metls, rgn iochr cn improve plnt growth especilly in sndy soil. However, the use of iochr is not without its critics. Reserch shows tht iochr cn contin dngerous inorgnic (hevy metls) nd orgnic contminnts (Hle et l., 1; Oleszczuk et l., 13; Buss nd Msek, 1; Domene et l., 15; Kołtowski nd Oleszczuk, 15). Where there re high levels of contminnts, there is risk of their uptke y plnts or migrtion down the soil profile to groundwter (Kołtowski nd Oleszczuk, 15). This my hve negtive effects for humns, for the environment nd for living orgnisms. Thus, iochr pplied to soils should e free of toxic sustnces efore ny future lrge-scle ppliction. The sld germintion test ws selected in the current study to test for slt stress where the iochr is mixed with n inert, finesnd medium nd the germintion of slt-sensitive species (sld, Lctuc stiv L.) is evluted. Thus, negtive effects t high ppliction levels on sld germintion my indicte high sh contents ut no hrmful sustnces. To test for toxic sustnces, the rley (Hordeum vulgre L.) germintion test ws selected where iochr is mixed into n orgnic medium (unfertilized pet sustrte) nd the plnt is not sensitive to slt stress. Thus, negtive effects t high ppliction levels on rley germintion my indicte the potentil toxic effects of hrmful sustnces produced during the process of pyrolysis. Mterils nd methods Sld germintion test The iochr ws mixed with n inert, fine-snd medium nd used in the sld germintion test sed on ISOe171 (Busch et l., 1), where the germintion of slt-sensitive species (Lctuc stiv L.) ws evluted. Severl different proportions of iochr nd fine snd were mixed following geometricl dilution. In ccordnce with the ISO test, the fctor is the mximl llowed, so the series used ws: % (control),.5%, 1%, %, % nd 8%. For one replicte, n mount of fresh weight equl to 1 g of dry weight ws tken out of the whole mixture nd filled into Petri dish. Tp wter ws dded to set the wter content to 85% of the mximum WHC. Forty seeds of Lctuc stiv L. were evenly sowed, leving free spce of round 1 cm to the order of the Petri dishes. The seeds were pressed softly into the sustrte. Afterwrd, 9 g (dry weight) of corse snd ws distriuted on top. The prepred Petri dishes were plced open in loted, zippered plstic g nd positioned in the green house. For the first 8 h, lck plstic foil covered the dishes. After 5 d incution, hrvesting commenced nd the numer of germinted seedlings nd the fresh nd dry weights of the ove ground iomss were determined. The Petri dishes, which still contined the soil mixture were mixed nd 5 g of dry weight were tken. Around 1 ml of wter were dded. After shking for 1 h t 15 rpm, the sustrte ws llowed to settle for 3 min nd then the ph nd electricl conductivity (EC) were mesured. All determintions were replicted three times. Brley germintion test Five iochr-pet mixtures were prepred, using smll mounts of iochr (%, 1%,.5%, 5% nd 1%) where 1% descries mixture of 1 g dry iochr with 99 g dry pet. Then, the dry weight nd wter holding cpcity (WHC) of the sustrtes were determined. To prepre 1 ml of dry weight for every mixture, the mount of fresh weight of iochr nd pet nd the mount of wter needed to set the mixture to % of the mximum wter holding cpcity (WHCmx) were clculted. Afterwrd, some textile/filter pper Germintion rte (%) Fresh weight per Petri dish (g) Fresh weight per plnt (mg) 5 3 1,5,,3,,1, CTR.5% 1% % % 8% Biochr (%) c CTR.5% 1% % % 8% Biochr (%) ws plced into the ottom of the plnt pots nd the mixtures were split into four replictes while leving sufficient to cover the seeds. After sowing seeds of rely in every pot, the rest of the mixture ws distriuted over the seeds nd the initil weight of the whole construction (pot þ mixture with % of WHCmx þ seeds) ws recorded. The plots were then rndomly plced in the greenhouse. Biochr (%) c CTR.5% 1% % % 8% (A) (B) (C) Fig. 1. Sld germintion test results: (A) germintion rte; (B) fresh weight per Petri dish; (C) fresh weight per plnt. Vlues re men þ SD of the three replictes. Different letters indicte significnt differences (one-wy nlysis of vrince) etween different mixtures of iochr nd the control (CTR).

3 L. Bouqis et l. / Agriculture nd Nturl Resources 51 (17) 7e5 9 ph vlues efore plnting 1 8 (A) ph vlues fter plnting 1 8 (B) CTR.5% 8% Biochr (%) CTR.5% 8% Biochr (%) EC vlues efore plnting (C) EC vlues fter plnting c (D) CTR.5% 8% Biochr (%) CTR.5% 8% Biochr (%) Fig.. Effect of iochr ppliction on ph nd electricl conductivity (EC) efore nd fter germintion of sld: (A) ph vlues efore plnting; (B) ph vlues fter plnting; (C) EC vlues efore plnting; (D) EC vlues fter plnting. Vlues re men þ SD (n ¼ 3). Different letters indicte significnt differences (one-wy nlysis of vrince) etween different mixtures of iochr nd the control (CTR). The weight of ech pot ws recorded dily nd the difference from the initil weight ws mde up y dding tp wter. The consumption of wter (through evpotrnspirtion) nd the produced iomss were used to determine the wter use efficiency (WUE). After 9 d, the germintion rte, iomss fresh weight, dry weight, wter content nd WUE (mg dw/g H O) were determined. Sttisticl nlysis For the rley nd sld germintion tests, the effects of different iochr dditions on ll replicted mesurements were tested using one-wy nlysis of vrince. The significnce of differences mong tretment groups ws determined using the Tukey test. A result ws considered significnt t p <.5. All sttisticl tests were performed using the SigmPlot 11 softwre (Systt Inc.; Chicgo, IL, USA). Results Sld germintion test In ll of the five repeted tests (.5%, 1%, %, % nd 8% iochr; Fig. 1A), the iochr incresed the germintion rte of Lctuc stiv ut not significntly even t mixture with.5% iochr. The highest germintion ws oserved with the highest ppliction rte nd represented n incresed germintion rte of 5% compred to the control. Determining the fresh weight per Petri dish of L. stiv seedlings in the iochr test indicted tht iochr incresed the fresh weight in ll of the five repeted tests ut the increse ws only significnt with the three highest ppliction rtes (Fig. 1B). Biochr incresed the fresh weight y 7% in the mixture with 8% iochr compred to the control. Determining the fresh weight per plnt indicted tht iochr incresed the fresh weight in the four repeted tests while it ws nerly similr in the lowest (.5%) ddition. A significnt increse ws oserved with the % nd % iochr pplictions (Fig. 1C) y % nd 9%, respectively, compred to the control. Fig. A (efore plnting) nd B (fter plnting) show the ph of ech mixture from the highest nd lowest rtes of ppliction compred to the control. With higher iochr concentrtion, the ph efore plnting ws lrge enough to exceed the ph for the control cse while it incresed (ut not significntly) with the lowest ddition. The sme results were found fter plnting. Fig. C (efore plnting) nd D (fter plnting) show the EC mesured t the highest nd lowest rtes of ppliction compred to the control. With higher iochr concentrtion, the EC ws lrge enough to exceed the EC for the control. With.5% iochr, efore plnting, the EC incresed (ut not significntly), while it decresed fter plnting. The wter holding cpcities were.3 g H O per g soil (dry weight),.39 g H O per g soil (dry weight) nd.3 g H O per g soil (dry weight) in the %,.5% nd 8% iochr-snd mixtures, respectively. The iochr ppliction incresed the WHC y 7.9% in the.5% nd y 1.8% in the 8% compred to the control. Brley germintion test In the three repeted test runs (1%,.5% or 5% iochr-pet mixture), the iochr incresed the germintion rte of rley ut not significntly (Fig. 3A) while t the high level of iochr (1%), smll ut not significnt decrese in the germintion rte ws

4 5 L. Bouqis et l. / Agriculture nd Nturl Resources 51 (17) 7e5 Germintion rte (%) Fresh weight per pot (g) Dry weight per pot (g) 1 8 8,,5,,3,,1, CTR 1%.5% 5% 1% Biochr (%) oserved. Fig. 3B shows the fresh weight of rley seedlings in the four iochr tests compred to the control. The results indicted tht the iochr incresed the fresh weight of rley in three repeted tests (1%,.5% or 5% iochr-pet mixture); ut the lrgest positive effect ws oserved in the.5% iochr in which iochr incresed the fresh weight y 1% compred to the control. At the higher level, iochr decresed the fresh weight of rley seedlings, ut none of the iochr dditions resulted in significnt difference to the control. Fig. 3C shows the dry weight of rley seedlings in the four iochr tests compred to the control. In ll repeted test runs, the iochr hd positive effect on the seedling dry weight. It incresed the dry weight even with the lowest ppliction rte; ut none of the iochr dditions resulted in significnt increse. The CTR 1%.5% 5% 1% Biochr (%) CTR 1%.5% 5% 1% Biochr (%) (A) (B) (C) Fig. 3. Effect of rgn shell iochr on germintion of rley: (A) germintion rte; (B) fresh weight per pot; (C) dry weight per pot. Vlues re men þ SD of the four replictes. Different letters indicte significnt differences (one-wy nlysis of vrince) etween different mixtures of iochr nd the control (CTR). lrgest positive effect ws oserved in the.5% iochr where the iochr incresed the dry weight y 13% compred to the control. Argn shell iochr hd positive effect on the wter content (Fig. A) t three iochr-pet mixtures (1%,.5%, 5%) while it decresed the wter content t the higher level (1%). The lrgest positive effect ws oserved with the.5% iochr where the wter content incresed y 1% compred to the control ut none of the iochr dditions resulted in significnt difference compred to the control. In ll of the four repeted tests: 1%,.5%, 5% nd 1% iochr of the iochr-pet mixture (Fig. B), there ws n increse (ut not significntly) in the wter use efficiency even with mixture with 1% iochr. The lrgest difference ws oserved with the.5% ppliction rte where the iochr incresed the wter use efficiency y 11% compred to the control. The wter holding cpcities were 5.89% g H O per g soil (mixture) dry weight, % g H O per g soil (mixture) dry weight nd.39% g H O per g soil (mixture) dry weight in the 1%, 5% nd % iochrpet mixtures, respectively. Discussion Sld germintion test The results reveled positive effect of rgn shell iochr on the germintion rte of sld in ll iochr tretments. The sme result ws oserved for the fresh weight of seedlings which is more sensitive prmeter to the iotoxic sustnces in the iochr (Fig. 1B nd C). Even with 8% iochr, positive effect ws oserved. Argn shell iochr hs highly lkline ph, high EC, high contents of K, N nd Mg nd low contents of C nd hevy metls compred to the soil (Bouqis et l., 1). Previous reserch hs reveled tht the contents of wter solule K, C, N nd Mg incresed significntly with iochr ppliction ecuse of the higher content of the ctions contined in the iochr reltive to soil (Biedermn nd Hrpole, 13; Jung et l., 1; Wng et l., 1). Moreover, iochr increses the retention of minerl nutrients which decreses lixivition nd is likely to increse nutrient vilility in the long term (Lehmnn nd Rondon, ; Lehmnn et l., ; Sigu et l., 1). Furthermore, chnges in the nutrient nd cron vilility fter iochr ddition to soils my hve positive impct on microil undnce, rhizoil nodultion nd soil fun ecuse iochr contins mcro- nd micro-pores (Lehmnn et l., 11; Zhng et l., 1). Such chnges in soil fun my well hve effects on nutrient cycles, soil structure nd indirectly ffect plnt growth (Lehmnn et l., 11). Bsed on these studies, the current results (incresing germintion rte, fresh weight of iomss, ph nd EC) cn e explined y the presence of more wter-solule nutrients in the mixture which minly originted from the rgn shell iochr. Thus, mixing rgn iochr nd soil would fcilitte ction retention in the soil. The ppliction of iochr t n incresing rte significntly incresed the ph efore nd fter plnting. Similr to ph, iochr ddition t high level incresed significntly the EC of the mixture efore nd fter plnting. The high ph of rgn shell iochr indicted tht sustntil portion of the nutrient slts sequestered in rgn wste ws concentrted during the pyrolysis process. The sme results were reported y Singh et l. (1), Biedermn nd Hrpole (13) nd Wng et l. (1). Given its high ph vlue, the rgn iochr could contin high levels of oth mcro- nd micro-nutrient elements, which were concentrted in the iochr during the pyrolysis process. Collectively, the results indicted tht rgn shell iochr would e eneficil to soil t these levels nd will e useful to increse the ph of cidic soils. The iochr ppliction incresed the WHC y 1.8% t 8% compred to the control.

5 L. Bouqis et l. / Agriculture nd Nturl Resources 51 (17) 7e5 51 7,5 5, Wter content (g) 3 WUE (g) 1,5 1, 1,5 CTR 1%,5% 5% 1%, CTR 1%,5% 5% 1% Percentge of iochr Percentge of iochr Fig.. Effect of rgn shells iochr on wter content nd wter use efficiency (WUE) 9 d fter germintion of rley: (A) wter content; (B) wter use efficiency. Vlues re men þ SD (n ¼ ). Different letters indicte significnt differences (one-wy nlysis of vrince) etween different mixtures of iochr nd the control (CTR). An increse in the WHC with iochr ppliction hs een reported y Ael et l. (13), Co et l. (1) nd Wng et l. (1). Brley germintion test The germintion results of different mixtures (pet-iochr) were compred with the germintion results of the control (unfertilized pet, % iochr) where the fresh/dry weight ws the most sensitive prmeter to indicte ny negtive effect of iochr on seedlings. In ll iochr tretments, no negtive effects of rgn shell iochr were oserved on the germintion rte of rley (Fig. 3A). The sme result ws oserved for the more sensitive prmeters of the fresh nd dry weight of seedlings to iotoxic sustnces in iochr (Fig. 3B nd C). No negtive effect of penut hull iochr on the germintion of rley ws reported y Busch et l. (1). Furthermore, rgn shell iochr hd positive effect on the germintion rte, seedling fresh/dry weight, wter content nd wter use efficiency in 1%,.5% nd 5% iochr-pet mixtures (Figs. 3 nd ) with increses in ll the prmeters, even t low levels of iochr tretment. At the highest level (1% iochr-pet mixture), iochr incresed the dry weight of the iomss nd the wter use efficiency. However, t 1%, iochr decresed (ut not significntly) the germintion rte nd fresh weight of rley seedlings compred to the control. The reduction of fresh weight could e ttriuted to delyed germintion tht could hve resulted in reduced time for growth (9 d) nd so led to reduced fresh weight. Delyed germintion ws lso reported for rley seeds from different iochrs (Brgmnn et l., 13). Thus, detiled reserch utilizing long-term experiments will e logicl next step. Compring the chemicl properties of rgn shells to the pet, there ws high content of K, N nd Mg nd low content of hevy metls; the oserved increses, even though low, minly in the fresh nd dry weights of iomss could hve een the results of the high level of wter solule nutrients nd the sence or low content of hrmful sustnces in the mixture with iochr ppliction. Thus rgn shell iochr could provide potentilly etter plnt growth. Argn shell iochr hs lso positive effect on the wter content nd wter use efficiency (Fig. A nd B) with increses in these two prmeters even t the lowest ppliction rte, lthough none of the iochr dditions resulted in significnt increse. As with the present study, improvement in the wter retention cpcity fter iochr ddition hs previously een oserved (Co et l., 1; Oi et l., 1) nd reported to e due to its porous nture (Downie et l., 9; Yu et l., 1). Furthermore, Lird et l. (9); Sun nd Lu (1) nd Oi et l. (1) hve reported tht the use of iochr s soil mendment ws nticipted to increse oth nutrient nd wter retention nd therey crop productivity where there is less dringe which ws in line with the current oservtions. Biochr use s soil conditioner is currently n importnt topic of reserch ecuse of the enefits to crop yield nd to the soil, y incresing the wter holding cpcity, ph, soil orgnic mtter levels nd fvoring microil ctivity. However, iochrs cn contin toxic compounds produced during the process of pyrolysis or ccumulted in the feedstock used in the production of iochrs. Thus, for future lrge-scle ppliction of iochr, it is importnt to ensure tht it will neither show toxic effects nor otherwise pose short- or long-term thret to soil nd the environment. To determine the potentil impct of toxic sustnces nd slt stress, this study focused on the effect of rgn shell iochr hs on seed germintion. Two different germintion tests were selected: 1) rley germintion test nd ) sld germintion test. The results otined from the two phytotoxicity tests reveled no negtive effects of rgn shell iochr on germintion, which provides preliminry indiction tht it could e sfely used for griculture. However, future reserch in long-term experiments will e logicl next step nd should consider chnges in nutrient vilility nd plnt growth. Conflict of interest None. Acknowledgements This work ws funded y the Hssn II Agronomic nd Veterinry Institute, Agdir, Morocco. The uthors would like to cknowledge Mr Mohmed Doulhousne for his contriution to the experimentl work. References Ael, S., Peters, A., Trinks, S., Schonsky, H., Fcklm, M., Wessolek, G., 13. Impct of iochr nd hydrochr ddition on wter retention nd wter repellency of sndy soil. Geoderm, 183e191. Bdroui, M., Agni, M., Soudi, B.,. Evolution de l qulite des sols sous mise en vleur intensive u Mroc. Conference Intensifiction gricole et qulite des sols et des eux, Rt, Mroc, 3 Novemre.

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