Relationships between Chemical Characteristics and Phytotoxicity

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Relationships between chemical characteristics and phytotoxicity of biochar from poultry litter pyrolysis Alessandro Girolamo Rombolà, Giovanni Marisi, Cristian Torri, Daniele Fabbri, Alessandro Buscaroli, Michele Ghidotti, and Andreas Hornung J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01540 • Publication Date (Web): 07 Jul 2015 Downloaded from http://pubs.acs.org on July 12, 2015

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Journal of Agricultural and Food Chemistry

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O

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poultry litter

corn stalk

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Relationships between chemical characteristics and phytotoxicity of biochar from poultry litter pyrolysis Alessandro G. Rombolà*§, Giovanni Marisi§, Cristian Torri§, Daniele Fabbri§, Alessandro Buscaroli§, Michele Ghidotti§, Andreas Hornung# §

C.I.R.I. Energia e Ambiente c/o Laboratory of Environmental Sciences “R. Sartori”/C.I.R.S.A., University of Bologna, via S. Alberto 163, 48123 Ravenna, Italy #

Fraunhofer Institute for Environmental, Safety, and Energy Technology UMSICHT, Institute Branch Sulzbach-Rosenberg (Germany)

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ABSTRACT: Three biochars were prepared by intermediate pyrolysis from poultry litter at

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different temperatures (400, 500 and 600 °C with decreasing residence times) and compared with

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biochars from corn stalk prepared under the same pyrolysis conditions. The phytotoxicity of these

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biochars was estimated by means of seed germination tests on cress (Lepidium sativum L.)

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conducted in water suspensions (at 2, 5 and 40 g/L) and on biochars wetted according to their water

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holding capacity. While the seeds germinated after 72 hours in water suspensions with corn stalk

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biochar were similar to the control (water only), significant inhibition was observed with poultry

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litter biochars. In comparison to corn stalk, poultry litter generated biochars with a higher content of

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ash, ammonium, nitrogen and volatile fatty acids (VFAs) and a similar concentration of polycyclic

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aromatic hydrocarbons (PAHs). Results from analytical pyrolysis (Py-GC-MS) indicated that

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nitrogen-containing organic compounds (NCCs) and aliphatic components were distinctive

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constituents of the thermally labile fraction of poultry litter biochar. The inhibition of germination

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due to poultry litter biochar produced at 400 °C (PL400) was suppressed after solvent extraction or

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treatment with active sludge. A novel method based on solid-phase microextraction (SPME)

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enabled the identification of mobile organic compounds in PL400 capable to be released in air and

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water, including VFAs and NCCs. The higher phytotoxicity of poultry litter than corn biochars was

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tentatively attributed to hydrophilic biodegradable substances derived from lipids or proteins

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removable by water leaching or microbial treatments.

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KEYWORDS: biomass, char, ecotoxicity, manure, VOC, pyrolysis.

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Biochar is the carbonaceous product of biomass pyrolysis which can be used as soil additive

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capable to mitigate a variety of agro-environmental stresses through the permanent storage of

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biomass carbon, pH correction, reduced synthetic fertilizer use, decreased runoff of fertilizers and

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agrochemicals.1-4

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However, the effects of biochar are highly variable depending on the feedstock, thermochemical

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process conditions, application rate, soil characteristics, environmental conditions, and plant species

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that are invoked to explain the variety of outcomes reported in literature that range from a boost in

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plant productivity to evident phytotoxicity.5,6

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Poultry litter is a waste biomass that has been investigated as a substrate for pyrolysis in the

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preparation of biochar.7-13 Poultry litter can be directly applied in agricultural soil as source of labile

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N, P and organic C, but drawbacks could be caused by excessive fertilisation and leaching.12 The

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conversion of these elements into slower cyclic forms by means of carbonisation can have potential

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benefits such as reduced loss of nutrients,13 improved nutrient availability7,8,11,13 and mitigation of

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greenhouse gas emission (CO2 and N2O).8 Priming and liming effect due to poultry litter biochar

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with increased N mineralisation has been described.10 The elimination of potential pathogens is an

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additional advantage advocating the use of a thermally treated poultry litter.8,12 Moreover, the

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production of biochar can be integrated with the generation of energy from renewable resources.12

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However, the use of biochar especially from animal origin has raised concerns related to its possible

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toxicity and studies have been recently conducted to explore physiological effects on biota.14-16

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Bioassays based on seed germination and early stage seedling growth are a simple and commonly

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used ecotoxicological tests for evaluating the impact of biochar amendment on crop growth.17 The

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biochars phytotoxicity test was made in absence of soil due to the large soil–char interactions

INTRODUCTION



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observed in some studies18 and because Solaiman et al.17 demonstrated that growing seedlings in

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pure biochar materials is a valid tool in assessing the effect of biochar application rate on

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germination.

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Seed germination, one of the most important phases in the life cycle of a plant, is highly responsive

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to existing environment.19 Factors such as heavy metals,20 PAHs,21 ammonia,22 salts23 and low

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molecular weight fatty acids24 have been shown to be responsible for inhibitory effects.

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Some studies have examined the effect of biochar on seed germination.17,25,26 Rogovska et al.21

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reported that biochars contain phytotoxic compounds that inhibit germination of maize. In contrast,

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Free et al.25 reported that maize seed germination was not significantly affected by biochars made

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from a range of organic sources. Solaiman et al.17 showed that biochars generally increased

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germination at low application rates (10-50 t/ha), whereas higher application rates of 100 t/ha had

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no effect or decreased germination rate. Alburquerque et al.27 observed that different biochars

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exerted a positive effect on seed germination also to high application rates instead.

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Recent studies have also suggested different methods for reducing the toxicity of biochar.28,29

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Washing biochar with water or an organic solvent has been successfully tested to reduce

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phytotoxicity of biochar.21,28,30 Meanwhile, Kołtowski et al.31 demonstrated significant reduction of

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biochars toxicity by drying them at various temperatures (100–300 °C) for 24 h.

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While the published and ongoing investigations are providing increasing data helpful to understand

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the relationships between biochar characteristics and seed germination,28,31 further studies are

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needed to better clarify the role played by the chemical properties in determining the toxicity effect

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on plant in order to forecast strategies in biochar synthesis or post-treatments. Biochars from

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different feedstock and process conditions may exhibit a wide range of plant response, from growth

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inhibition to stimulation. The relatively simple seed germination test is a valid and fast tool to

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compare several biochars obtained from different starting materials and under different pyrolysis

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conditions. Since the test is performed in short time and without the buffering effect of soil, it could


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be considered a kind of precautionary procedure that highlights intrinsic phytotoxicity of the tested

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materials.

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The aim of this study was to evaluate phytotoxicity of biochar from poultry litter by means of

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standard germination tests and to identify possible relationships with its chemical characteristics. To

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this purpose, germination tests with cress (Lepidium sativum L.) were conducted to poultry litter

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biochars synthesised at different pyrolysis conditions. Besides manure, poultry litter typically

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contains bedding such as sawdust, wood shavings, rice hulls and straw.8,12 Therefore, a comparison

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was made with biochars produced from a lignocellulosic material, prepared under the same

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conditions, in order to better discriminate the contributions between lipids/proteins and

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(hemi)cellulose/lignin. The effect of solvent extraction and biological conditioning on seed

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germination was tested on a selected poultry litter biochar prepared upon pyrolysis at 400 °C

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(PL400). The chemical composition of mobile constituents in this sample capable to be potentially

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released in the water and air compartments was investigated by solvent extraction and solid-phase

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microextraction (SPME).

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Biochar synthesis. Cornstalk was described in a previous study.32 Granular poultry litter was a

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marketed organic fertilizer obtained after processing raw poultry litter collected from local broiler

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farms by pasteurizing at 80–110 °C, milling, and pelletizing. Biomass was air dried at 60 °C, milled

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and sieved at 2 mm before pyrolysis.

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Batch pyrolysis experiments were conducted under nitrogen flow with a fixed bed tubular quartz

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reactor placed into a refractory furnace (see Conti et al.33 for details) with about 20 g cornstalk or 35

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g poultry litter exactly weighed and uniformly placed onto a sliding quartz boat; nitrogen flow was

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set at 1500 cm3/min and when the temperature inside the reactor, measured with a thermocouple,

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reached the selected value, the boat was pushed into the oven and left for a given residence time

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before pulling it back into the unheated part of the reactor. Pyrolysis were performed under three

MATERIALS AND METHODS



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different conditions based on a previous study33 of temperature/residence time: 400 °C/20 min, 500

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°C/10 min and 600 °C/5 min. In accordance to the original biomass (cornstalk, CS; poultry litter,

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PL) and pyrolysis temperature, the obtained biochar samples were named CS400, CS500, CS600,

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PL400, PL500 and PL600. Chemicals were purchased by Sigma Aldrich. SPME Carboxen-PDMS

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fibers and the fiber holder were purchased by Supelco.

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Biochar Characterization. Elemental composition (HCNS) was determined by combustion using a

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Thermo Scientific Flash 2000 series analyzer. Ash was determined as the residual mass left after

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exposure at 600 °C for 5 hours. The oxygen content was calculated from the mass balance:

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O%=100-(C+H+N+ash)%.

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Analytical pyrolysis (Py-GC-MS) were conducted at 900 °C for 100 seconds with a CDS 5250

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pyroprobe interfaced to a Varian 3400 GC-Saturn 2000 MS. GC-MS conditions and the

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determination of indicators of carbonisation % charred and toluene/naphthalene ratio were

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described in Conti et al.33

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The content of the 16 EPA priority PAHs was measured in triplicate as described in Fabbri et al.34

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Briefly, about 0.5 g of biochar were spiked with 0.1 mL of surrogate PAH mix (Supelco for EPA

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525 containing acenaphthene-d10, phenanthrene-d10 and chrysene-d12 5 µg/mL each in

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acetonitrile) and soxhlet extracted with acetone/cyclohexane (1:1, v/v) for 36 hours. The solution

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was filtered, added with 1 mL of n-nonane (keeper), carefully evaporated by rotatory vacuum

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evaporation at 40 °C and cleaned up by solid phase extraction onto a silica gel cartridge before

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analysis with a Agilent HP 6850 GC coupled to a Agilent HP 5975 quadrupole mass spectrometer;

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GC-MS conditions were those detailed in Fabbri et al.34 Recovery of surrogate PAHs was

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determined with respect to the internal standard tri-tert-butylbenzene added prior to GC-MS

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analysis.

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Volatile fatty acids (VFAs) were determined by the single drop extraction procedure as described in

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Torri et al.35 About 200 mg of biochar exactly weighed was added with 0.1 mL of internal standard

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solution (1 g/L 2-ethylbutyrate in deionised water) and thoroughly mixed with 0.2 mL of saturated 5 Environment ACS Paragon Plus

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aqueous KHSO4. After centrifugation, a drop of dimethyl carbonate (1.2 µL) from a 10 µL

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chromatography microsyringe was exposed into the supernatant aqueous solution. After 20 min

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exposure the drop was retracted and injected into a GC-FID (injection temperature 250 °C)

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equipped with polar GC column (Agilent Q7221J&W nitroterephthalic-acid-modified polyethylene

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glycol DB-FFAP 222 30 m, 0.25 mm, 0.2 µm) with the following thermal program: 80 °C for 5

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min, then 10 °C/min to 250 °C. Calibration was performed by applying the same procedure to

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standard solutions containing known concentration of each VFA (namely: acetic, propionic,

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isobutyric, butyric, isovaleric and valeric acids).

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For the determination of N-NH4+, about 10 g of biochar were placed in an end-to-end shaker for 2 h

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with 1 N KCl (1:10 dw:v) followed by centrifuging at 4500 x g for 20 minutes and passing through

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a 0.45 µm paper filter.

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SPME of mobile compounds. An aliquot (1mL) of the aqueous extract solution was introduced

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into a vial mixed with 0.5 mL of KH2PO4/Na2HPO4 phosphate buffer 2M at pH 5.3, then spiked

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with o-eugenol 1µg/mL and 2-ethyl butyric acid 5 µg/mL as internal standards. SPME analyses

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were performed by directly exposing the Carboxen-PDMS fiber into the test solution under

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magnetic stirring. After 30 minutes exposure, the fiber was inserted into the injector of an Agilent

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5977 gas chromatograph and analytes were thermally desorbed at 250 °C for 10 minutes and

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analysed as described for VFAs with a DB-FFAP polar column (30 m lenght, 0.25 mm i.d, 0.25 µm

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film thickness). Detection was made with a quadrupole mass spectrometer Agilent 7820A operating

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under electron ionization at 70eV with acquisition at 1scan/sec in the m/z 35 and 450 range. Mass

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spectra were acquired in full scan mode with a gain factor of 5. Prior to sample analysis, blanks

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were performed first with a thermal desorption of the fiber alone and then after a 30 min exposure

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to a solution of deionized water with 0.67 M phosphate buffer.

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Head space (HS) analysis was performed following the procedure described by Spokas et al.36 (150

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°C, 30 minutes) but using SPME instead of syringe sampling. Poultry litter biochar was weighed

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(0.5 g) into 20 mL head space vials. The vials were spiked with 1µg of o-eugenol (1000 µg/mL 6 Environment ACS Paragon Plus


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solution in methanol) and then sealed. HS-SPME was carried out by placing the fiber holder at the

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top of the vial and exposing the Car-PDMS fiber to the headspace close to the cap (30 minutes),

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while bottom of the vial was put on a heating plate at 150 °C. GC-MS analysis was carried out as

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described above but with the GC oven starting from 36 °C held for 5 minutes. Prior sample

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analysis, blanks were carried out first with a thermal desorption of the fiber alone and a sealed vial

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with empty headspace.

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Biochar post-treatments. Aqueous extraction. About 2 g of PL400 was extracted with 50 mL of

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deionised water in a 100 mL flask at room temperature for 12 hours with mechanical shaking. The

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aqueous phase was separated by filtration through a 0.22 µm paper filter and used as such for the

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germination test, while an aliquot was kept at -20 °C for SPME-GC-MS analysis (see above). The

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solid biochar residue left after water extraction was further extracted with 50 mL of methanol under

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reflux for 12 hours. The methanol was separated by filtration and an aliquot corresponding to the 40

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g/L suspension of biochar was poured into petri dishes and dried overnight at 70 °C under vacuum

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to remove all the methanol. Thereafter deionised water was added to perform germination tests. The

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final solid biochar residue left after water and methanol extraction was dried overnight at 100 °C

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under vacuum and utilized for germination tests.

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Biochar post-treatments. Biological treatment. Microbial treatment of PL400 was conducted

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with an activated sludge obtained from a municipal wastewater treatment plant located in Ravenna

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after centrifugation at 6000 rpm (20% w/w volatile suspended solids). A suspension containing 0.5

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g of the concentrated sludge and 250 mL of 40 g L-1 PL400 in deionised water was thoroughly

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mixed under laminar shake at 120 rpm overnight. An aliquot of 10 mL of this suspension was added

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in petri dishes and stored for 14 days at 25 °C before performing germination test as shown above.

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Germination tests. The germination tests were conducted in four replicates by incubating 50 seeds

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of cress (Lepidium sativum L.) with 5 g of a mixture containing biochar and deionized water onto

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sterilized cellulose filter paper (Whatman No. 1) placed in a petri dish sealed with paraffin film.

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Three levels of biochar concentration were tested 2, 5 and 40 g/L. These rates were equivalent to 2, 7 Environment ACS Paragon Plus

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5 and 40 t/ha on an area basis of 10 cm soil depth and a dry bulk density of 1.5 kg/m3. Germination

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tests were also performed on the fractions obtained from the chemical and biological post-

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treatments of PL400 described above. The quantity of these fractions was adjusted to correspond to

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the concentration level of 40 g/L of the original biochar. Before incubation, the samples were

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shaken at 150 rpm on a platform shaker at room temperature for 24 h. pH and electrical

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conductivity (EC) were both determined. The pH was directly measured placing the glass-electrode

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into the suspension with a pH-meter Mettler Toledo SG 2-ELK. The EC was measured with a Delta

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OHM HD 8706 conductimeter in the supernatant obtained by centrifuging the suspension and

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filtered at 0.45 micron.

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Phytotoxicity tests were performed on biochar:deionized water mixtures (wetted biochar) according

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to the procedure described in UNI 11357:2010. The experiments were conducted with 50 seeds of

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cress which were incubated with 10 g of biochar saturated with deionized water according to value

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of the water holding capacity (Table 1) on sterilized cellulose filter paper placed in a petri dish. All

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Petri dishes were covered and incubated in room thermostat at 25 ± 2 °C for 72 ± 0.5 hours in the

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dark. Similarly, a control was prepared with deionised water.

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After 72 hours of exposure, a visible root development was used as the operational definition of

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seed germination. Data were reported as relative seed germination (RSG) percentage with respect to

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the control (deionised water):

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RSG = (number of germinated seeds in the sample/ number of germinated seeds in control) * 100.

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Statistical analysis. All the experiments were conducted at least in duplicate. Results of

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germination tests were evaluated statistically using Analysis of Variance (ANOVA) performed with

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R software version 3.1.2 (http://www.r-project.org) followed by Student-Newman-Keuls post hoc

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tests. The difference between the treated groups and the control group was evaluated with Dunnett’s

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t-test (p < 0.05).

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RESULTS AND DISCUSSION 8 Environment ACS Paragon Plus


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Characterization of biochar

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Bulk analysis. The yields and characteristics of biochars obtained from the pyrolysis of poultry litter

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(PL400, PL500, PL600) under three different pyrolysis conditions are reported in Table 1 and

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compared with those of biochar from corn stalk (CS400, CS500, CS600). As expected, the chemical

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characteristics of the biochars were dependent on the original biomass and the pyrolysis conditions.

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In particular, the H/C and O/C ratios decreased with increasing pyrolysis temperature, while the

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content of ash increased as observed with the same pyrolysis unit under the same conditions.33

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Biochar from poultry litter contained higher levels of nitrogen, sulphur and ash, derived from the

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manure fraction, as demonstrated by comparative studies on manure and lignocellulosic biochars.9

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In general, the elemental composition, ash content and the trends with pyrolysis conditions of the

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investigated poultry litter chars were comparable to those reported in the literature.7,8,37

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Extractable compounds. The concentrations of specific potentially toxic extractable compounds,

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namely PAHs, VFAs and ammonium are reported in Table 2. Solvent extractable PAHs occurred

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within the range of 0.7-1.7 mg/kg, values that were typical of biochars from different origins.34,38,39

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PAHs concentration can be considered negligible for acute effect lower than typical values in

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soils.40 Generally, naphthalene was the most abundant PAH followed by phenanthrene and fluorine;

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the level of benzo[a]pyrene was in the 5-65 ng/g range.

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The concentration of VFAs was significantly higher in poultry litter biochars (4-9 mg/kg) in

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comparison to those from cornstalk (2-4 mg/kg). Acetic acid was always the most abundant VFA

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and could be derived from the thermal degradation of hemicellulose.41 VFAs could also be derived

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from the thermal degradation of triacylglycerols42 and free fatty acids.43

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Ammonium was not detected in cornstalk biochar, whereas it was abundant in biochars from

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poultry litter with higher concentrations in the less carbonised biochars.

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Py-GC-MS. Thermolabile fraction. The molecular composition of the thermally labile fraction

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could be inferred from the structural identification of the compounds identified in the

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pyrolysates.33,34,44 The pyrolysate of CS400 was characterised by a complex pattern of compounds

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dominated by phenols and methoxyphenols associated to the presence of partially charred lignin,

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while the pyrolysate of CS600 contained few peaks due to the hydrocarbons associated to more

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heavily charred fraction (Figure 1). Proxies of the degree of carbonisation established in previous

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studies33 for lignocellulosic biomass were confirmed in this study for corn stalk biochar: the

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toluene/naphthalene ratio and the relative abundance of compounds representative of the charred

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fraction (% charred) exhibited a clear trend with H/C ratios (Table 2). However, biochar samples

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from poultry litter did not exhibit significant changes with the H/C ratios and the variability was

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higher. This finding would suggest that the pyrolysis proxies developed for lignocellulosic biochar

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could not be valid for biochar containing charred proteins and lipids.

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The occurrence of partially charred components from proteins and lipids were clearly evidenced in

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the pyrolysates of biochar. Phenols and methoxyphenols were detected in PL400 as well as in

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CS400 in accordance to the fact that the original substrates contained a lignocellulosic component.

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The distinctive signature of PL pyrolysate was the occurrence of nitrogen-containing compounds

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(NCCs) from proteins and a pattern of n-alkanes/n-alk-1-enes assigned to the thermal cracking of

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bound or free fatty acids (Figure 1). The occurrence of saturated alkyl domains was confirmed by

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in the availability of sorbed compounds.37 Among the tentatively identified NCCs, pyrrole,

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pyridine, (iso)quinoline and carbazole along with their alkyl derivatives are indicative of partially

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charred proteinaceous matter. It is worthwhile to note that NCCs were also identified in the volatile

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fraction by SPME as described in the next section.

C-NMR studies on poultry manure biochars that disappear after carbonisation and may play a role

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SPME-GC-MS. Volatile and water soluble compounds. Information on the molecular characteristics

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of the mobile fraction was gathered by HS-SPME (volatile) and DI-SPME (water soluble). Poultry 10 Environment ACS Paragon Plus


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litter contains heavy metals that may end up in the resulting biochar.45 However, our attention was

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specifically focused to organic compounds, being reported that heavy metals are generally present

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below the limits causing adverse effects and loosely bioavailable.46 Moreover, Bastos et al.16 argued

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that in aqueous extracts PAHs and metals might occur at concentrations below the level to pose

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detrimental effects, at least for woody biochar up to 80 t/ha; however, it has to be remarked that the

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biological response depends on the organisms selected in the bioassay,16 and the content of metals

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depends on pyrolysis conditions,45 thus the influence of inorganic constituents cannot be always

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excluded.

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SPME was applied to sample PL400 that was utilised in post-treatment studies. The results are

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shown in Figure 2 for the analysis of volatile organic compounds (VOCs) by HS-SPME (Figure

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2A) and DI-SPME of aqueous extracts (Figure 2B), respectively. The VOCs were characterised by

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the presence of a wide array of compounds deriving from the thermal degradation of

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polysaccharides (e.g. cyclopentenones, furans), lignin (e.g. 4-vinylphenol, guaiacol), proteins (e.g.

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pyrroles, pyridines, indole), lipids (e.g. VFAs, acetic acid is also derived from hemicellulose).

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Alkylated pyrazines and acetamide were probably derived from Maillard reactions between

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carbohydrates and proteins.

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Notably, a suite of short chain n-alkanes/alkenes were identified supporting Py-GC-MS results and

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literature data about the occurrence of aliphatic components in poultry litter biochar.37 It is expected

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that the polar fraction of VOCs will be preferentially distributed into the aqueous phase in

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comparison to non-polar constituents. In fact, the SPME-GC-MS analysis of the PL400 water

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extract showed a predominance of organic acids, including C2-C10 aliphatic and C7-C9 aromatic

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acids (Figure 2B).

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Effect of biochar on seed germination. The pH and EC of the biochar/water suspensions utilized

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in germination tests are reported in Table 3. The pH values were higher for the suspensions, from

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the same feedstock, with the more carbonized biochars and increased, for each biochar type, with


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increasing concentration. Under the same conditions, the pH was higher in the suspensions with

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poultry litter biochar, in accordance to previous studies.9

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The EC values of the suspensions increased with increasing biochar concentration and, at the same

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concentrations, the biochar from poultry litter had much higher (from 4 to 40 times for the same

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condition) EC values than the CS suspensions. Salinity can have a detrimental effect on seed

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germination and plant growth, especially in the seedling stage, though the response of various plant

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species to salinity differs considerably.47 In general, salinity effects are mostly negligible in extracts

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with EC readings of 2000 µS/cm or less.48 This critical level was exceeded in poultry litter biochar

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at 40 g/L. The toxicity of inorganic nitrogen results mainly from ammonia (NH3) which affects

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plant growth and metabolism at low concentration levels at which NH4+ is not harmful.47 At

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concentrations of 0.15-0.20 mM, which are comparable to those calculated in the 40 g/L

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biochar/water mixtures, NH3 could be toxic.49

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The effect of the biochar suspensions on seed germination of cress (Lepidium sativum L.) is

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presented in Table 4 in terms of relative seed germination percentage with respect to control

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(deionised water only).

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The assay results in this work suggested that all the cornstalk biochar suspensions had little impact

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on seed germination as one-way ANOVA analysis showed no significant difference between

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control group and test groups (p > 0.05). Noticeably, CS400 was almost non-toxic to germination

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even if used as the growth substrate (UNI test, Table 3).

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On the contrary, all the biochar samples from poultry litter inhibited significantly the seed

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germination at the highest level of 40 g/L in water suspensions. At the harsh conditions of the UNI

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test the germination was totally suppressed.

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The comparison with cornstalk suggested that the toxicity of biochar from poultry litter could be

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explained by some distinctive chemical components originated from this feedstock. Compounds

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derived from lignin and cellulose/hemicellulose could be excluded on the ground that biochar

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samples from corn stalk did not suppress seed germination in water suspensions. The biochars 12 Environment ACS Paragon Plus


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suspensions from both substrates presented similar pH values, thus this parameter is not involved in

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toxicity. This is in accordance to Gell et al.50 findings who did not evidenced clear trends of pH and

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short term phytotoxicity in biochars of different origins, at least under neutral/basic conditions.

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Similarly, the solvent extractable PAHs concentration was similar in PL and CS biochars, thus

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PAHs cannot be responsible of the observed toxicity. The calculated concentrations of VFAs in

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biochar suspensions (from data of Table 2) are at levels that may cause detrimental effects, for

320

instance calculated PL400 VFAs at 40 g/L (374 µg/g) was higher than 252 µg/g EC50 values for

321

plant growth.46 However, different factors are governing the physiological response of VFAs

322

including pH and bio-availability.51,52Acetic acid was present in all the biochars, partly due to the

323

decomposition of cellulose/hemicellulose. In addition, the occurrence of benzoic acid and other

324

aromatic acids was identified by DI-SPME along with VFAs. Benzoic acid is reported to inhibit

325

hydraulic conductivity and nutrient uptake by plant roots, thus resulting in growth inhibition.53

326

However, no significant effect of benzoic acid on Orobanche crenata seed germination was

327

observed by Fernández-Aparicio et al.54

328

The suite of alkanes/alkenes characterising the Py-GC-MS pyrolysates of poultry litter biochar

329

samples would suggest the presence of a lipid fraction producing shorter chain fatty acids by

330

thermal degradation as confirmed by SPME-GC-MS on PL400.

331

The main differences between the CS and PL biochars were the higher content of elemental

332

nitrogen (Table 1) and ammonium (Table 2), and the presence of a thermally labile fraction derived

333

from proteins and lipids (Py-GC-MS data).

334

Germination tests after biochar post-treatment. A selected sample of poultry litter biochar

335

(PL400) was extracted with water followed by methanol extraction; the extracts and the residue

336

were utilized in germination tests. Germination tests were also performed to PL400 after treatment

337

with sewage sludge to assess the effect of biodegradation. The quantity of extracts and the residues

338

corresponded to the biochar loading level of 40 g/L. The results are presented in Figure 3. The cress

339

germination rate in the water extracts was similar to that of the original biochar suspensions 13 Environment ACS Paragon Plus

Page 14 of 28

Page 15 of 28


340

indicating inhibition due to some components in the water extracts. The germination rates increased

341

significantly to values similar to the control when the suspension was made with the biochar left

342

after solvent extraction. These observations are supported by the results of Rogovska et al.21, who

343

showed that growth inhibition no longer occurred when biochars were washed prior to germination.

344

Biochar suspensions treated with an active sludge for almost two weeks displayed a germination

345

rate similar to the extracted biochar (Figure 3). The reduced toxicity could be ascribed to microbial

346

degradation of some noxious components as suggested by Bargmann et al.28

347

The results showed in Figure 3 indicated that the relative seed germination of water extracts is low

348

and comparable to that of the original biochar strongly supporting the hypothesis that the polar/ionic

349

constituents ending up in water are responsible to the observed biochar toxicity. Similarly,

350

Bargmann et al.28 applying germination tests to hydrochars from various origin demonstrated that

351

the inhibiting effects were caused by some water soluble substances. These authors hypothesized

352

that organic acids could be possibly responsible of the water extractable fractions toxicity. The

353

potential of microbial detoxification was evidenced by Busch et al.26 who observed that the

354

genotoxicity of hydrochar mixed with compost became lower than that of pure hydrochar.

355

Gell et al.50 showed that the short term phytotoxicity of biochar is dependent on the feedstock and is

356

probably associated to ionic water soluble constituents rather than the less polar organic compounds

357

composing tars. In accordance, the methanolic extract of biochar after water extraction exhibited a

358

seed germination rate of 93% (not reported in Figure 3) higher than that measured in the water

359

extracts. Interestingly, among the various biochars investigated by Gell et al.50 those obtained from

360

poultry biochar exhibited positive effects (radish root elongation) and acted in decreasing

361

phytotoxicity of digestates.

362

The results of this study suggested that toxic compounds responsible for the toxicity of PL400 were

363

water extractable and biodegradable. The SPME analysis of the water extracts (Figure 2) evidenced

364

that aliphatic and aromatic carboxylic acids were the dominant compounds. Py-GC-MS and HS-



365

SPME analyses evidenced the presence of NNCs that seemingly were not partitioned into the water

366

phase.

367

Biochar has a potential as a soil amendment for improving soil quality, decrease fertilizers losses

368

and store carbon into the soil. Nevertheless, as soil additive, the absence of phytotoxicity is the

369

minimal requirement. Biochar from poultry litter may exert negative effect at least at the relatively

370

high level of soil amendment (40 t/ha) due to the presence of water soluble and biodegradable

371

components, probably derived from the thermal decomposition of proteins and lipids. However, the

372

toxicity can be drastically reduced by means of washing with water or mixing with biologically

373

active material. Whereas leaching (accompanied by wastewater generation) would be not an

374

applicable option, biological treatment (e.g. composting or mixing with activated sludge) of

375

phytotoxic biochars could be a simple and economic solution for increase the agronomic

376

performance of biochar characterized by toxicity issues. Results obtained shows that biochar are not

377

an “intrinsically safe” material, and every biochar (from different process and/or feedstock) has to

378

be evaluated, checked and eventually treated before the agronomic application.

379 380

381

Corresponding Author

382

*Alessandro G. Rombolà. Fax: +39 0544 937411. E-mail: [email protected]

383

Notes

384

The authors declare no competing financial interest.

AUTHOR INFORMATION

385 386

387

This study was partly conducted within the framework of the APQ Ricerca Intervento a “Sostegno

388

dello sviluppo dei Laboratori di ricerca nei campi della nautica e dell’energia per il Tecnopolo di

389

Ravenna” “Energia, parte Biomasse” between Università di Bologna and Regione Emilia Romagna

390

(Italy). The authors acknowledge Denis Zannoni University of Bologna for ammonium analysis.

ACKNOWLEDGMENTS


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Page 17 of 28


391

Part of this study was based on the master thesis by Giovanni Marisi at the University of Bologna

392

Ravenna Campus. The SPME study was conducted in the framework of the Fraunhofer UMSICHT

393

- University of Bologna Collaboration Agreement.

394 395 396 397 398 399 400 401 402 403

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TABLES AND FIGURE

Table 1. Yields, water-holding capacity, elemental analysis and ash (% wt dry weight mean values ± s.d. n=4) and elemental molar ratios of biochars from the pyrolysis of corn stalk (CS) and poultry litter (PL) at different conditions (400 °C/20 min, 500 °C/10 min and 600 °C/5 min).

Yield (% wt)

waterholding capacity (%)

CS400

38.3±0.9

CS500

Biochar

Elemental content (%)

Ash (%)

Molar ratios H/C

C

H

N

O

S

69.5

50±2.1

3.3±0.1

0.96±0.03

15±1.9

0.07±0.01

28.95±0.01

0.79

33±1.6

81.1

51±1.6

2.7±0.1

0.91±0.04

14.9±0.1

0.03±0.04

30.14±0.02

0.63

CS600

31.4±0.5

73.7

50.7±0.3

2.4±0.1

0.81±0.03

13±1.6

-

32.30±0.02

0.57

PL400

49±3.4

88.6

33±4.7

2.7±0.5

3.6±0.8

11±2.1

1.7±0.5

46.64±0.01

0.98

PL500

41.4±0.9

94.1

33±1.0

2.1±0.1

3.4±0.1

6.5±0.9

2.2±0.1

52.29±0.04

0.76

PL600

39.5±0.5

92.3

31.4±0.5

1.7±0.1

3.2±0.5

4.6±0.3

2.3±0.1

56.82±0.01

0.65



Page 24 of 28

Table 2. Molecular analysis of extractable compounds and volatile matter by Py-GC-MS of biochar from corn stalk (CS) and poultry litter (PS) (mean values and s.d. from two replicates, T/N toluene/naphthalene ratio). Extractable Biochar

PAHs

VFAs

Py-GC-MS NH4+

mg/kg mg/g mg/kg CS400 0.72±0.06 3.8±1.2

% charred 53±3

T/N 8.5±0.7

CS500 1.09±0.05 0.9±0.3

-

80±3

5.4±1.5

CS600 0.84±0.01 2.6±0.1

-

92±3

3.0±1.7

1.7±0.2

9.3±0.3

45

88±9

13±10

PL500 0.88±0.05 4.3±2.0

25

90±3

12±6

PL600 0.79±0.01 6.8±1.8

14

88±3

11±2

PL400

Table 3. Results from chemical analysis of biochars and relative water suspensions.

Biochar

Electrical conductivity (mS/cm)

pH 2 g/L 5 g/L 40 g/L

2 g/L

5 g/L 40 g/L

CS400

7.6

8.0

8.5

16

75

1.8 103

CS500

8.2

8.5

9.3

72

76

1.9 103

CS600

8.4

8.9

10.1

1.9 102 3.5 102 1.9 103

PL400

8.0

9.0

9.7

7.1 102 1.3 103 7.3 103

PL500

8.4

9.3

10.2

8.7 102 1.5 103 7.7 103

PL600

9.4

9.8

10.3

9.3 102 2.0 103 8.1 103


Page 25 of 28


Table 4. Relative seed germination (% of control) of cress (Lepidium sativum L.) in biochar:deionized water suspensions (2, 5, 40 g/L) and phytotoxicity tests (% seed germination with respect to control) according to UNI 11357:2010 (mean values and s.d. from four replicates). The percent seed germination in pure deionised water (control) is also reported. *Indicates differences at the 0.05 level compared with the control by Dunnett’s tests. Germination (%)

Biochar Id. Control

2 g/L 96±2

5 g/L 97±2

40 g/L 98±2

UNI 94±2

CS400

98±2

93±1*

96±4

81±3*

CS500

97±5

98±1

96±2

41±3*

CS600

95±4

95±2

96±1

14±4*

Control

92±2

92±2

92±2

94±1

PL400

83±4*

74±3*

52±19*

no germination

PL500

77±2*

73±2*

47±10*

no germination

PL600

77±3*

74±3*

53±7*

no germination



Page 26 of 28

Figure 1. Total ion chromatograms from Py-GC-MS of poultry litter (PL) and cornstalk (CS) biochars.

OH

C2

MCounts 20

N H

PL 400

CN OH

CH3 C2

N N N H

10

n-alkanes/n-alkenes

CH3

N H

PL 500

5.0 N H

PL 600

N

1.0

N H

5

10

15

20 OH

C2

MCounts

25

30 min

35

OH

OH

OCH3

CH3

CS 400 OH OCH3 OCH3 OH

OH

5.0 C2

OCH3

CS 500 O

5.0

CS 600 O

5.0

5

10

15

20

25

30 min


35

Page 27 of 28


Figure 2. Total ion chromatograms obtained after (A) HS-SPME of volatile organic compounds and (B) DI-SPME of water extract of poultry litter biochar (PL400).

O O

Counts

O OH

OH

OH

O

2.6e+07

MeO

OH

2.4e+07

i.s.

O

2.2e+07

B

OH

O OH

i.s.

2e+07

OH

O

1.8e+07

OH

O

1.6e+07

OH

O

1.4e+07

O

COOH

OH

1.2e+07

OH O

1e+07

COOH OH

8e+06

O

6e+06

COOH

OH

4e+06 2e+06 10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

26.00

28.00 Minutes

OH

N

O

N

6e+07

i.s.

C5

A

C2

S

5.5e+07

O O

O

N

S

5e+07 4.5e+07

MeO

CHO

O

Counts

OH

C2

O

O

C6-C10 hydrocarbons

N N

4e+07

O

O

N

NH2 N

3.5e+07 3e+07

OH

O

2.5e+07

O

OH C3

OH

2e+07 1.5e+07 1e+07 5e+06 2

4

6

8

10

12

14

16

18

20

22

24


26

28

Minutes


Figure 3. Seed germination rates relative to control of original poultry litter biochar (PL400), water extracts and PL400 after post-treatments (solvent extraction and treatment with active sludge). Germination tests referred to a biochar load of 40 g/L. The germination rate of control (water only) is reported for comparison. *Indicates differences at the 0.05 level compared with the control by Dunnett’s tests.


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Relationships between Chemical Characteristics and Phytotoxicity

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