Characterization and ecotoxicological investigation of biochar produced via slow pyrolysis: Effect of feedstock composition and pyrolysis conditions

Accepted Manuscript Title: Characterization and ecotoxicological investigation of biochar produced via slow pyrolysis: effect of feedstock composition and pyrolysis conditions Authors: Xiao Yang, Weicheng Ng, Belinda Shu Ee Wong, Gyeong Hun Baeg, Chi-Hwa Wang, Yong Sik Ok PII: DOI: Reference:

S0304-3894(18)30956-7 https://doi.org/10.1016/j.jhazmat.2018.10.047 HAZMAT 19868

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

19-2-2018 9-10-2018 15-10-2018

Please cite this article as: Xiao Yang, Weicheng Ng, Belinda Shu Ee Wong, Gyeong Hun Baeg, Chi-Hwa Wang, Yong Sik Ok, Characterization and ecotoxicological investigation of biochar produced via slow pyrolysis: effect of feedstock composition and pyrolysis conditions, Journal of Hazardous Materials https://doi.org/10.1016/j.jhazmat.2018.10.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Characterization and ecotoxicological investigation of biochar produced via slow pyrolysis: effect of feedstock composition and pyrolysis conditions

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Xiao Yang1,2†, Weicheng Ng3†, Belinda Shu Ee Wong4, Gyeong Hun Baeg4, Chi-Hwa Wang3,*, Yong Sik Ok1,** 1

Korea Biochar Research Center, O-Jeong Eco-Resilience Institute (OJERI) & Division of

Environmental Science and Ecological Engineering, Korea University, Seoul, Republic of Korea 2

Department of Biological Environment, Kangwon National University, Chuncheon 24341,

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Republic of Korea

Department of Chemical and Biomolecular Engineering, National University of Singapore,

The authors contributed equally to this work.

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Medical Drive, Singapore 117594

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Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4

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Singapore 117585, Singapore

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Corresponding Authors:

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* Prof. Chi-Hwa Wang, Tel: +65 6516 5079, Fax: +65 6779 1936, E-mail: [email protected] ** Prof. Yong Sik Ok, [email protected] Highlights   

Toxicity of biochars was studied using fruit flies and human cell lines. Heavy metal (HM) & polycyclic aromatic hydrocarbons (PAHs) level was insignificant. The biochars had limited impact to the viability of fruit files. 1

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The biochars inhibited the growth of cells but unlikely caused by HMs and PAHs. Surface area of biochar may help to capture toxicants and reduce bioavailability.

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Abstract: This study systematically investigated the biochar toxicity from the in vitro tests involving the use of human liver and lung cell lines, as well as in vivo tests using Drosophila melanogaster (fruit fly). Biochars used in this study were produced from vegetable waste, pine cone and their mixture

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(1:1 by weight) at two representative temperatures (200 and 500 oC). Two common toxicant groups

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in biochar, heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) contents, were

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detected for clarification of the relationship between their toxicity behaviors and biochar bulk

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characteristics. The results showed that 1) no HMs can be found in the biochar if HMs are absence in their feedstock 2) PAHs were formed during the pyrolysis no matter what type of biomss used,

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but the concentration is low that can be acceptable for soil legislative criteria 3) biochars had

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limited impact to the viability of flies, but inhibited the growth of the cells 4) the low leaching potential of HMs and PAHs (total 16 USEPA) in the studied biochars may not be the major reason

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which put the harm to the cell, more effort on the identification need to be done. This work can

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provide a new picture to the biochar researchers for better understanding of the two faces of biochar.

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Keywords: biochar; toxicity assessment; pyrolysis; polycyclic aromatic hydrocarbons.

1. Introduction 2

Biochar, the technologically so-called charred carbon-rich residue produced by a controlled heating process, has been studied extensively over the last decade [1]. The interesting characteristics of biochar, such as high external surface area, consisting of inorganic nutrient (N,

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P, K etc.), modifiable surface and structure and environmental stability, allow for wide application of biochar in terms of soil remediation, water treatment, and energy production as well as catalysis [2–4]. Compared to other carbon materials, biochar production technology has many advantages, such as commercially available starting materials, mild reaction conditions (<700 °C, low energy inputs, and atmosphere requirements), and easy to operate (minimal equipment requirements) [5].

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After delving deep into the study of biochar, researchers began to realize biochar’s contradictory

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nature. Despite its environmental benefits, biochar itself could be a carrier of contaminants, either

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due to the improper use of hazardous feedstocks (heavy metals (HMs) and metalloids) or the

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unexpected by-products (toxic polycyclic aromatic hydrocarbons; PAHs) generated during the pyrolysis [6]. The concentration of these toxic substances may not be very significant, but the harm

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from such exposure is great. Therefore, it is imperative to establish a rapid toxicity screening

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platform for biochars before their massive uses in various applications [7]. The contaminants

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inherent to biochar will be introduced into the environment during biochar application. Moreover, the cumulative emission of contaminants could cause negative effects and pose health risks to

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humans.

Studies on the assessment of biochar intrinsic toxicity have been carried out recently. Hale et

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al. [8] determined the profiles of total bioavailable PAHs and dioxins in several biochars. They concluded that compared to slow pyrolysis biochars, fast pyrolysis biochar and gasification biochar contained relative higher total PAH concentrations, ranged from 0.3 to 45 µg g-1, which is above warning limits by some regulations. Oleszczuk et al. [9] assessed the toxicity of biochar produced 3

from biogas residue. They reported that a variety of HMs (Cr, Cd, Cu, Pb and Mn) and PAHs were co-produced during the char formation and the composition of feedstock and pyrolysis conditions determined the type and concentration of the toxic materials. The ecotoxicological results showed

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that when biochar was added to the soil, the biochar-induced toxicants inhibited seed germination and root growth of Lepidium sativum. However, these studies only focused on the biochar produced from a single-feedstock and its effect on soil quality and plant growth. Many studies have advocated for the use of mixed-feedstock for biochar production and claimed that using mixed feedstock for biochar production could effectively improve the nutritional value, tune the

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structural features, and reduce the economic costs of the biochar [8–10]. Because mixed feedstock

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biochars are potential tools in agricultural and environmental management activities, it is

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imperative to study their toxicological effects to evaluate whether they are acceptable to

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environmental concerns. Moreover, when considering low density and fine powder forms, biochar fine particles would be diffusely involved in different media and potentially cause some

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environmental concerns, i.e. resuspension in the atmosphere, deposition and accumulation in the

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river sediments, diffusion and leaching in the surface water or even penetrate into the ground water

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via “colloid-facilitated transport”[11–13]. Therefore, the unknown attributes of biochar severely constrain the development of biochar technology.

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Above all, to fill in the important gaps in knowledge, the focus of this study was determination of potential toxic materials that exist in biochar by considering the contents of HMs and metalloids

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and the 16 priority PAHs recognized by United States Environmental Protection Agency (USEPA). Biochars were synthesized from vegetable waste, pine cones and a mixture of the two (1:1 by weight) via a slow pyrolysis. To provide a rapid means of studying the toxicity of the biochars, in vitro tests involving the use of human liver and lung cell lines, as well as in vivo tests using 4

Drosophila melanogaster (fruit fly), were conducted in similar manners as those in authors’ previous work [14]. Biochar-carried contaminants would unavoidably enter the environment, in particular when in contact with rainfall and surface water [15]. The potential entrance of biochar

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leaching toxins into the blood circulatory system and subsequently to the liver when humans consume food or water contaminated with toxic biochar or its leachate justifies the investigation employing liver cells. Meanwhile, the risk of breathing in fine particles when handling biochar is the motivation for the study using lung cell lines. Monitoring the changes of cell viability assay in response to biochar leachate could help researchers to build up a rapid standard platform for

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evaluating biochar toxicity. Drosophila melanogaster is also a commonly used and good model

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for studying toxicity, owing to its fast process (short life cycle of fruit flies), cost-effectiveness,

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and, more importantly, high genomic similarity to humans [16]. Solid biochar at different

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concentrations was spiked into fly food and the number of progenies eclosed over 7 days was used as a viability/ toxicity indicator. For human cells, the leachate of biochar at different concentrations

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was exposed to the cells for different periods, up to 72h, and the cell viability was measured.

2. Materials and Methods

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2.1 Biochar production

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Biochar feedstocks, i.e., vegetable waste and pine cones, were collected respectively from a grocery market and Kangwon National University (KNU) in Chuncheon, Gangwon-do, Korea.

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The raw materials were first dried in a greenhouse and then placed into an air-blowing thermostatic oven at 50 °C to achieve constant weight. The composition of the waste was analyzed (Table S1). Before pyrolysis, the dried samples were ground and sieved to less than 2mm. Then, the biomass was transferred into an alumina crucible and heated in a muffle furnace (LT, Nabertherm, Germany). The peak temperature was set to 200 or 500 °C at a ramp of 7 °C/min, and kept for 2h. 5

The pyrolysis was conducted under hermetic conditions, without oxygen compensation. A mixed feedstock biochar was obtained by heating a mixture of the vegetable waste and pine cones at a 1:1 weight ratio. More details are provided in a previous study by authors [10]. Six produced

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biochars were labeled as V200, V500, P200, P500, PV200 and PV500, which V represents vegetable waste, P represents pine cones, PV represents the mixture of the two and numbers represent the processing temperature.

2.2 Metals and metalloids analysis and PAHs determination of the biochars

Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to detect the

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presence of metal(loid)s (if any) in the biochars. Solid biochar samples were subjected to acid

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digestion using 3:1 HNO3/HCl, microwaved at 180°C, and finally topped up to 10mL with

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deionized water. Any precipitate observed was filtered prior to ICP-OES analysis. The Dual-view

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Optima 5300 DV ICP-OES system, which has a minimum detection level of 0.1 mg/L, was used. The concentrations of 20 metal(loid)s of interest, i.e. Ag, As, Ba, Ca, Cd, Co, Cr, Cu, K, Mn, Mo,

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Na, Ni, Pb, Sb, Se, Ti, Tl, V and Zn, were examined. The determination of 16 PAHs in biochar

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specimens followed the national standard method of China (HJ 784-2016) with modifications.

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Details have provided in SI.

2.3 Toxicity assessment via Drosophila melanogaster test and cell viability assays study

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Drosophila melanogaster was used to study the toxicity of six types of biochar at different dosages, i.e. 0, 1.5, 3 and 5 mg/mL. The protocol was similar to that in a previous study [14]. The

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emission of biochar-derived contaminants to the environment is mainly through the leaching when in contact with water [15]. The biochar leachate was prepared for assessing its potential toxicity. A modified USEPA toxicity characteristic leaching procedure was used [17]. Biochar was leached at liquid-to-solid (L/S) ratio of 10, and then MRC-5 and HepG2 cells (from American Type Culture 6

Collection (ATCC), USA) were exposed at different concentrations of the leachate of each studied biochars. Cells culture and analytical methods were displayed in SI. 2.4 Statistical analysis

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Mean values with standard errors were used. Data processing for multiple comparisons, including one-way analysis of variance (ANOVA), significant difference test (p<0.05) and Pearson correlation between the biochar properties and biochar-induced toxicity response was performed using IBM SPSS Statistics 23.

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3. Results and Discussion

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3.1 Biochar characterization

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Proximate analysis, measurement of the main four ingredients of the biochar, was carried out based on the description in the previous work [18]. As shown in Table1, high temperature biochar

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displayed higher resident matter and ash, but lower mobile matter in comparison to low

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temperature biochars. Higher temperature favors the development of the resident matter, also known as fixed carbon, indicating highly condensed and thermally stable components were formed

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during the pyrolysis [19]. In addition, the biochar yield dramatically decreased when the pyrolysis

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temperature increased from 200 to 500 °C. The thermal decomposition of lignin and cellulose could have been responsible for this reduction in yield. As temperature rises, organic compounds

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inside raw biomass break down into small gaseous molecules, such as CO, CO2, CH4 and H2, which are released from the matrix, resulting in biochar weight loss [20]. Resident matter is often considered an index of biochar resistance to the environment, serving as a primary source of reference for C sequestration when the biochar is returned to the soil [21]. Based on the compositional variation in feedstock, the biochar derived from the herbaceous-rich biomass 7

contained less resident matter compared to the lignin-rich biomass, which ranged from 25.77-50.17 wt.% for V biochars and 35.60-79.60 wt.% for P biochars [22]. There are reports that the abundant resident matter in biochar makes it stable to the aging process and microbial degradation, while

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the mobile matter acts as a C resource, supplying organic nutrients to the soil and “food” to the microorganisms [23,24]. However, the trend in ash content was the opposite, showing that biochar derived from V had higher ash content than that of P biochar. The results demonstrated that the characteristics of the mixed-feedstock biochar lie between those of biochars derived from the single feedstocks.

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In the ultimate analysis, all biochars had high C content, particularly in the case of the high

(IBI;

http://www.biochar-

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Initiative

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temperature products. According to the regulation promulgated by the International Biochar

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international.org/sites/default/files/Guidelines_for_Biochar_That_Is_Used_in_Soil_Final.pdf), all the biochars produced at 500 °C, excluding V500, were assigned to class 1 (total C ≥ 60%). The

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others were graded into class 2 (30% ≤ total C ≤ 60%). High pyrolysis temperature biochar turned

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out to have higher values of C and N, and lower values of H and O regardless of the feedstock type

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used. Thermal-induced dehydration and decarboxylation reactions have been known to account for these changes [25]. The relative contents of nonvolatile, including minerals and non-

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combustion substances, could have been concentrated owing to the overall weight reduction caused by volatiles release. The H/C and O/C ratios were calculated to assess the biochars’

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aromaticity and polarity, respectively [4]. It has been reported that when the H/C value of biochar is below 0.3, the biochar is thought to having a highly condensed structure, whereas a value greater than 0.7 represents that a non-condensed structure [26]. P500 had the lowest H/C value, followed by PV500 < P200 < V500 < PV200 < V200. This suggested that the stable aromatic ring system 8

formed more easily in samples that underwent high temperature pyrolysis. Although the type of raw material influenced, in part, the aromaticity of the biochar, the sample produced from cellulose-rich biomass had weaker stability than the one from lignin-rich biomass. Similar to the

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H/C trend, a low molar O/C ratio was observed in 500 °C pyrolyzed biochars compared to those pyrolyzed at 200 °C, indicating that increasing the pyrolysis temperature facilitated the formation of a less hydrophilic surface [27]. Interestingly, when the pyrolysis conditions (500 °C under limited O supply) were equal, the mixed-feedstock biochar had a lower O/C ratio compared to those of V500 and P500, suggesting the most hydrophobic biochar surface generated in PV500. In

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other words, the use of mixed-feedstock for biochar production not only adjusted the elemental

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composition, but also tuned biochar’s surface chemistry. This was further supported by the analysis

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of pH, EC and surface physical properties. For example, biochar produced from V at 500 °C had

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a basic surface (pH>11) and high EC value, which may account for the concentration of alkali salts in the ash content. However, the pH and EC were relatively low in the case of P500. Thus, the

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addition of vegetable waste to pine cones can increase the pH and EC of the pyrolyzed product of

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such. Furthermore, P500 possessed an advantage in aspect of microporosity, thus addition of pine

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cones can effectively increase the external surface area of V biochar. Therefore, utilization of a mixed-feedstock for biochar production is a valid option for tailoring the biochar physico-chemical

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properties to address specific environmental concerns and application requirements.

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3.2 Analysis of toxicants in the biochars The contents of the two most common contaminant sources in biochar, i.e., HMs and

metalloids, were determined through ICP analysis and that of PAHs via HPLC analysis. Table 2 presents the analyzed HM concentrations of each biochar. It was found that none of the six biochars exhibited significant levels of HMs. Only alkaline and alkaline earth metals, such as K, Na and Ca 9

were present at higher percentages, as they are minerals commonly observed in many biochars though, they may be present in varying amount depending on the type of feedstock used [28–30]. It is noteworthy that the HM levels in biochar are mainly dependent on the feedstock composition.

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If the raw material is naturally free of HMs, the derivative biochar contains no HM contents. However, biochar production techniques are double-edged in that pyrolysis not only boosts the useful minerals but also concentrates the harmful HMs. Therefore, screening for the appropriate feedstock, that is one with low or no HMs, prior to pyrolysis is a critical prerequisite for biochar production to ensure its safe application. The absence of toxic HMs in studied biochars is hence a

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good indication that the biochar can be safely used in numerous applications [2,5,31–33].

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Since PAHs are highly pathogenic for humans, as they have carcinogenic and mutagenic

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characteristics, USEPA has labeled them as priority contaminants and issued a list of 16 PAHs in

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1976 to standardize the most concerning set of compounds for assessing risks, particularly in the environmental sciences. PAHs appear widely in biochar products owing to the incomplete

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combustion of organic materials in biomass as well as condensation and aromatization of solid

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materials during biochar formation [34,35]. Fig. 1 shows the representative composition of the 16

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PAHs in different biochars. The results showed that differences in the total content of 16 PAHs content in biochars were mainly due to the treating temperature and composition of the feedstock,

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ranged from 330 to 6930 µg/kg. Based on the recommended limits issued by Germany’s Federal Soil Protection (Basic quality scenario), all the tested biochars met the requirements and were

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below the threshold (<12000 µg/kg) [36]. However, P200 (6930 µg/kg) and PV500 (3823 µg/kg) approached or even exceeded the premium quality scenario regulated by Switzerland’s Chemical Risk Reduction Act [36]. Although the testing values differ from the extraction method applied for PAH quantification, the trend could be identified. The lowest levels of the 16 PAHs were found in 10

V biochars. Generally, evolution of PAHs often occurs in high temperature pyrolyzed samples, which probably be attributed to the re-condensation of PAHs sorbed on the biochar surface [30] . It’s worth noting that the greater BET surface area is, the more extractable PAHs can be found.

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The exception was for P biochars, as a decrease was noted in the total levels of 16 PAHs. Above all, the total 16 PAHs in studied biochars were relatively low and acceptable for soil remediation purpose.

With respect to the fractionation of PAHs content in the 500 °C pyrolyzed biochars, the leading fraction was 2-ring naphthalene, followed by 3-ring acenaphthene and acenaphthylene, and 4-ring

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chrysene. The concentrations of 5- and 6-ring PAHs in most of the biochars were below the

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detection limits, though trace amount of 5-ring benzo[b]fluoranthene was determined in P200 and

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P500. The low temperature pyrolysis process may have led to the formation of PAHs, irrespective

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of feedstock effect, with the ranking as 3-ring > 2-ring > 4-ring > 5-ring. No 6-ring PAHs were found in any of the biochars. In all investigated biochars, the 3- and 4-ring PAH levels decreased

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with increasing pyrolysis temperature. Accordingly, the values of H/C and O/C decreased, which

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agrees with the results published previously [39]. However, the opposite trend emerged for the

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case of 2-ring PAHs. Their contents increased as the temperature rose, except for pine cones case. Another factor that influences the biochar PAH content is the intrinsic properties of the biochar’s

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feedstock. A high level of PAHs was detected in P biochars, which was significantly higher than that of the cases without its involvement, whatever the species of PAH. Only a limited amount of

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USEPA PAHs was detected in biochar made from woodchips, pea straw, and vegetation via slow pyrolysis, which ranged from 0.01 to 0.12 mg/kg [40]. The values observed in this study were significantly higher than those values by 2.6 to 4.6 times, particularly when P were used as the pyrolysis precursor. Instead, our records are more like the values of gasification biochar produced 11

from chestnut and bamboo, which were reported to have 1.01-3.68 mg/kg of USEPA PAHs found in their experiment [41]. Additionally, the concentration of the sum of 16 PAHs in PV200 was situated between those of P200 and V200. It should be stressed that unlike the biochar generated

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from single feedstock, PV500 displayed a different pattern than that of biochar derived from the single feedstock, presenting the highest level of 2-ring naphthalene. Some unknown interactions between the two feedstocks during the pyrolysis might have happened, and thus led to the enhancement of naphthalene in the pyrolytic products. Therefore, it was concluded that 1) the total USEPA PAH concentration, to a large extent, depends on the 2- and 3-ring PAHs, which contribute

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approximately 70-95 % of total PAHs; 2) the proportion of heavy PAHs (5- and 6-ring PAHs), or

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highly toxic fractions, was extremely low, that is, in most of cases below the threshold of detection;

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3) although biochar contains a certain amount of PAHs, the values remain below the warning limits

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set by the European Soil Society; and 4) improper combination of raw biomass for biochar

hindering its development.

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production may result in a rise in PAH content, thereby, posing challenges to the environment and

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3.3 Drosophila melanogaster toxicity study

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Effects of the biochars on the growth of Drosophila melanogaster were investigated by considering the dosage and type of biochar as well as the pyrolysis temperature. Fig. 2 shows the

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number of flies hatched and the viability of the fruit flies after exposure to different biochars at

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different concentrations. At the dosage of 1.5 mg/mL, the application of single-feedstock derived biochar slightly enhanced the viability of flies, and there were no significant differences amongst these treatments. However, a negative response was noted for the mixed-feedstock derived biochar. When the dosage increased to 3 mg/mL, fly viability was improved by the PV biochar; it was even superior to the biochars produced from the P and V individually. As the biochar dosage increased 12

to 5 mg/mL, limited impact on the fly viability occurred. Noted that biochar produced at high temperature led to higher fly viability than the one produced at low temperature; in particular, V500 and PV500 increased the viability by 8% and 13%, respectively. In general, the application

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of biochar at low dose offered a slight enhancement of fly growth, except for PV500, which positively affected fly viability at high dose. In general, it was found that application of biochar at concentrations between 0 mg/mL and 5 mg/mL had minimal effect on the bioactivity of fruit flies, regardless of the source of biochar feedstock type (vegetable waste, pine cones, or their mixture) or pyrolysis temperature (200 and 500 °C). The results of toxicity test could be used to explain this

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observation. On one hand, no significant level of toxic HMs was present in the biochar, most of

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HMs were even below the detection limit. Since the bioavailability of PAH contents would vary

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with the testing method, the concentration level of the tested 16 PAHs was not high based on the

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employed method (HJ 784-2016). Due to different bio availabilities, solid matter content is an insufficient indicator for toxicity observed in leachates. Henceforth, the leaching potential of the

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detected PAHs in biochars might cause toxicity to living organisms.

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3.4 Cell viability assays for toxicity evaluation of the biochar leachates

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Apart from the investigation of biochar toxicity to living organisms, which have a complete immune system, cell experiments were also carried out because of its high sensitivity to the

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hazardous materials. Fig. 3a-3c shows the viability of MRC-5 cells (human lung cells) after exposure to the various biochar leachates. Concerning the time-dependent effect for a given

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biochar leachate concentration, generally the cell viability did not differ notably for 24, 48, nor 72 h of exposure time. Regarding the dose-dependent effect, it was observed that none of the biochar leachates at the lowest concentration tested (C/C0=0.01) resulted in significant cell death. As the concentration increased, the biochar leachates, excluding the P500, for which increasing the 13

concentration up to C/C0=1 still led to about 100% cell viability, displayed decreasing cell viability to different extents. Of the six biochar leachates, that of V200 caused the most cell death and hence gave the lowest cell viability. Even at C/C0=0.1, it gave ~20% viability, whereas the others were

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still at approximately >80% viability. Additionally, it was also not surprising that the cell viability of PV200 was somewhere between that of P200 and V200, whereas the cell viability of PV500 was somewhere between that of P500 and V500, owning to the mixing of two types of biochar. The biochar toxicity towards the MCR-5 cells depended predominantly on the composition of the raw biomass used for biochar preparation and pyrolysis conditions. When improper feedstock was

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employed, the derivate biochar negatively affected cell growth. Incorporation of harmless biomass

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(pine) during pyrolysis alleviated the pathogenicity to the cells somewhat, but failed to eliminate

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it. Previous studies stated that increasing temperature inhibits the formation of toxicants in the

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subsequent biochar, especially for organic compounds such as PAHs, therefore reducing their environmental threat [38]. However, this did not apply to the V biochars (C/C0=1), as the mortality

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of MCR-5 was not reduced by elevating pyrolysis temperature. This indicated that, in some cases,

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the pyrosynthesized toxicants in V biochar have a certain resistance to heat.

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The results of viability assay using HepG2 cells are shown in Fig. 3d-3f. In general, the exact same trend and pattern as those of MRC-5 cells were observed. The only exception was that

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because HepG2 is a liver cancer cell line, it was stronger and more resistant to the toxicants, hence, its viability was higher when compared to MRC-5 cells. One distinct difference was with V200 at

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C/C0=0.1, where a significant drop in viability was observed for MRC-5 cell line, but not for HepG2 cell line due to its higher resistance. It was also noted that, for both cell lines, some cell viabilities were slightly greater than 100%, especially for low concentrations leachates (C/C0=0.01, and in some cases, C/C0=0.1). While high toxicity causes excessive reactive oxygen species (ROS) 14

production and cell death, relatively low toxicity produces low levels of ROS, which may actually promote cell proliferation via alterations in cell signaling, rather than cell death [42,43]. 3.5 Characteristics versus toxicity behavior of the biochars

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Further investigation via Pearson correlation between the biochar quality parameters and corresponding toxicity response in both fly and cell studies was done and the results are presented in Table 3. Based on ICP analysis of metal(loid)s of biochars, as presented in Table 2, no significant levels of toxic HMs were detected. The alkali metals (Ca, K) in biochars, reflected by salinity (EC), have no remarkable effect in relation to the toxicity. Therefore, the decrease in cell viability

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observed here likely was not due to HM toxicity, but rather may be attributed to some other factors.

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One of the main reasons postulated was the mobile matter (MM) content in the biochars and their

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leachates. The concentration of MM in biochars was negatively related to the toxicity behavior of

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the biochars. This phenomenon correlated well with the results reported in previous experimental

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works, in that high MM in biochar could potentially inhibit plant growth, reduce plant nitrogen

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uptake and induce microbial growth [44,45]. Furthermore, low temperature biochar will generally have higher MM content [10,45]. Therefore, when comparing V200 and V500, it was noted that

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V200 which was produced at a lower temperature caused more cell death (more “toxic”) than its V500 counterpart produced at higher temperature. A similar trend was observed for P200 and P500,

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as well as for PV200 and PV500. This postulation was further supported by the results in our

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previous work [10] where the microbial community ratios (in terms of total fatty acid methyl ester (FAME)) in soils treated with these six types of biochar were investigated. In that study, it was found that low temperature biochars (200 °C) had higher percentage of MM (~50-60% MM) than that of high temperature biochars (500 °C) (~10% MM), and that low temperature biochars increased the soil microbial activity due to the supplement of readily available carbon. V200 was 15

found to be the most effective in increasing microbial activity (highest total FAME value), while P500 was the least effective (lowest total FAME value). These microbial community results are consistent with the current cell viability data. Nevertheless, it should be noted that though low

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temperature biochars caused cell death, they could in improve soil quality of contaminated soils by immobilizing cationic HMs and, increasing the soil microbial community abundance and biogeochemical reactions [10].

Some studies have connected biochar toxicity to biochar-containing PAHs, stating that unfavorable effect on the viability of F. candida and germination of L. sativum should lie behind

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more PAHs formed in high temperature pyrolyzed biochar [46,47]. However, the biochar-induced

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toxicity towards to living organism has no univocal conclusion. In this study, no significant link

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was found between the growth inhibition of tested living organism and biochar-carried PAHs at

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what species and which concentration. Moreover, the molar ratios of H/C and O/C were proposed as an index of biochar toxicity. Along with the increment of carbonization, thermal decomposition

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of organic matter in biomass rendered the decrease in H/C and O/C. Meanwhile, level up the

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proportion of carbon, that are, resident matter and elemental carbon, therefore enable the biochar

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has more condensed and aromatic π−π bond [7]. Nevertheless, deepening pyrolysis reaction could strengthen the biochar microporosity and lead to higher surface area and reduced toxicity (r=0.987,

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P<0.01 for MCR-5; r=0.932, P<0.01 for HepG2). These changes offered a chance for boosting biochar adsorption capacity, and hence, occluded and locked up the toxic material within biochar

A

matrix which effectively reduced the emission of pyrosynsized toxicants. The results provided strong evidence for this argument, though the sort of toxicants that induced the biotoxicity remains unclear. One possible cause of such toxicity might be pyrosynthesized volatile organic compounds (VOC), dioxins or even PAH species out of the US EPA list. Research has testified biochar products 16

invariably contain certain VOC contents no matter what type of pyrolysis condition and biomass used. Future experimentation to determine the profiles of the toxin fractions in biochar is extremely

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important to arm people for better management of biochar technology.

4. Conclusions

Biochar intrinsic toxic material associated with their impact to the living organism need to be taken into consideration when such a material was applied. This study built up a rapid operative and accurate protocol to examine the ecotoxicity of biochar to ensure use safety. It can be

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concluded that 1) toxicants could be generated when converting the biomass into biochar via

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pyrolysis, no matter what types of biomass were applied; 2) the selection of safe feedstock and

A

optimization of pyrolysis conditions are critical for the control of toxicants profile in a biochar; 3)

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the concentration of biochar-containing PAHs content are relative low and acceptable from the

D

related environmental legislative standpoint; 4) biochar had limit impact to the viability of flies

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but negatively affected the growth of human liver and lung cell at high concentration exposure; 5) although previous studies reported that high level of HMs and PAHs had a potential harm to the

EP

plant growth and soil biota, this work found that the low leaching potential of HMs and PAHs (total 16 USEPA) in studied biochars may not be the major reason for the cell growth inhibition.

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The observed cell growth inhibition may be caused by unknown toxic materials released from

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biochar matrix, so more work should be performed to identify the species of biochar-induced hazardous materials in the next stage. Nevertheless, increasing biochar surface area favors to reduce biochar toxicity because all the intrinsic toxicants will be captured by the porous structure.

Acknowledgements 17

This research programme is funded by the National Research Foundation (NRF), Prime Minister’s Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) programme with Grant Number R-706-001-101-281, National University of Singapore.

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The authors thank Xiaoqiang Cui for his comment on the interpretation of section 3.5 and Drs. Xu Zhen, Pooya Davoodi and Anbu Mozhi Thamizhchelvan on the clarification of LOQ unit for heavy metal detection.

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Table 1 Selected characteristics of the studied biochar samples. Adapted from reference [12].

5.95 11.23 4.15 6.77 5.26 10.39

dS m-1 0.041 0.121 0.001 0.001 0.000 0.045

Mobile Resident Ash C H O N Matter Matter ---------------------------------------- % ---------------------------------------75.76 1.21 56.44 25.77 16.59 41.05 5.35 27.96 3.26 29.28 0.72 12.43 50.17 36.67 48.21 1.99 10.85 3.49 86.53 1.28 62.35 35.60 0.77 62.50 1.91 24.28 0.93 35.78 1.42 10.02 79.60 8.96 73.11 2.56 20.51 1.77 79.59 1.00 58.37 32.72 7.91 49.79 5.38 35.39 0.52 33.85 1.07 10.33 70.53 18.06 67.81 2.19 7.87 3.00 Yield Moisture

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EC

H/C

O/C

1.56 0.49 0.42 0.37 1.30 0.39

0.51 0.17 0.21 0.29 0.53 0.09

BET Surface Pore area volume m2 g-1 m3 kg-1 0.36 2.59 1.16 2.42 0.47 2.38 192.97 10.2 0.44 0.43 50.26 3.22

Pore size nm 43.24 22.80 45.13 2.44 23.27 54.61

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CC E

PT

ED

V200 V500 P200 P500 PV200 PV500

pH

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Sample

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Table 2 ICP analysis of the metal(loid)s of the solid biochars. Unit for metal concentration: µg/kg. Limit of quantification (LOQ): 100 µg/kg. “ND” stands for “not detected”. V, P, and PV stand for vegetable waste, pine cones and mixture of the two, respectively. Numbers stand for pyrolysis temperature. V200 V500

P200 P500 PV200 PV500

ND ND <100 8900 ND ND <100 <100 39800 100 ND 4200 ND ND ND ND 100 ND ND <100

ND ND <100 600 ND ND ND ND ND <100 ND 200 ND ND ND ND <100 ND ND ND

ND ND <100 8800 ND ND ND <100 53900 <100 <100 9300 ND ND ND ND <100 ND ND <100

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ND ND <100 3000 ND ND ND ND 12800 <100 ND 3000 ND ND ND ND <100 ND ND ND

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ND ND <100 2800 ND ND <100 <100 400 <100 ND 400 <100 ND ND ND <100 ND ND <100

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M

ND ND <100 22300 ND <100 <100 <100 75600 200 ND 9500 ND ND ND ND 200 ND ND <100

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TE

D

Ag As Ba Ca Cd Co Cr Cu K Mn Mo Na Ni Pb Sb Se Ti Tl V Zn

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Table 3. Pearson product moment correlation coefficients (r) between biochar quality parameters and viability of flies and cells. Test Conditions subject FV 5 mg/L MRC-5 48 h, C/C0=1 HepG2 48 h, C/C0=1

Mobile Resident matter matter

EC

Ca

K

-.004 -.366 -.446

-.040 -.329 -.448

.071 .467 .466

.836* .747 .821*

C

H/C

O/C

SA

2 rings PAHs

3 rings PAHs

4 rings PAHs

.029 -.252 -.376

.792 .706 .900*

-.623 -.427 -.589

-.724 -.114 -.354

.670 .987** .932**

.684 .072 .446

-.350 -.260 -.119

-.181 -.108 .040

5 rings Total PAHs PAHs -.012 .129 .006 -.113 .170 .191

A

CC E

PT

ED

M

A

* Correlation is significant at the 0.05 level ** Correlation is significant at the 0.01 level FV: Fly viability; SA: Surface area.

-.730 -.505 -.508

Ash

26

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TE

D

M

A

N

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Fig.1 Total concentrations of the 16 US EPA PAHs and the sums of different ring number PAHs determined for the different biochars. V, P, and PV stand for vegetable waste, pine cones and mixture of the two, respectively. Numbers stand for pyrolysis temperature.

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Fig.2 Viability of fruit flies after exposure to different types of biochars at different concentrations (1.5, 3 and 5mg/mL). V, P, and PV stand for vegetable waste, pine cones and mixture of the two, respectively. Numbers stand for pyrolysis temperature.

27

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(c)

(e)

(f)

A

CC E

PT

(d)

ED

M

A

(a)

Fig. 3 Viability of MRC-5 cells (a,b,c) and HepG2 cells (d,e,f) after exposure to different concentration of biochar leachates for 24, 48 and 72 h. V, P, and PV stand for vegetable waste, pine cones and mixture of the two, respectively. Numbers stand for pyrolysis temperature. Different letters above the vertical bars indicate statistically significant differences at p < 0.05 (Tukey's HSD test).

28