Ecotoxicology and Environmental Safety 107 (2014) 306–312
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Assessment of mutagenic potential of pyrolysis biochars by Ames Salmonella/mammalian-microsomal mutagenicity test Reshma Anjum n, Niclas Krakat, M. Toufiq Reza, Michael Klocke Leibniz Institute for Agricultural Engineering Potsdam-Bornim e.V., Max-Eyth-Allee 100, D-14469 Potsdam, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 23 March 2014 Received in revised form 3 June 2014 Accepted 4 June 2014 Available online 20 July 2014
Biochar is of raising interest in sustainable biomass utilization concepts. Particularly biochar derived from pyrolysis attaches important agricultural capacities mandatory for an improved carbon sequestration, soil fertility and amelioration, respectively. In fact, large scale field trials and commercial business with biochar materials have already been started but still only few are known about the mutagenic potential of biochars produced. In this study hemp bedding and wood pellet biomass were used for biochar production by pyrolysis. The total concentrations of polycyclic aromatic hydrocarbons (PAHs) were 34.9 mg g 1 of dry mass and 33.7 mg g 1 of dry mass for hemp biochar and wood biochar, respectively. The concentration of PAHs in tar produced during wood carbonization was 17.4 mg g 1. The concentrations of phenolic compounds were 55 mg g 1 and 8.3 mg g 1 for hemp and wood biochar, respectively. Salmonella/microsomal mutagenicity tests (i.e. Ames test) revealed a maximum mutagenicity for hemp biochar extracts with strains TA97, TA98 and TA100 in the presence and absence of liver microsomal fractions, respectively. Wood biochar and tar extract exhibited maximum mutagenicity with strains TA98 and T100 both in the presence and absence of liver microsomal fraction. The reversion of the applied tester strains increased in the presence and absence of liver microsomal fractions with an increasing dose of hemp biochar extract up to 2 ml per plate and decreased at a concentration of 2.5 ml per plate. For wood biochar and tar extracts, reversion of tester strains increased both in the presence and absence of S9 at extract concentrations of 4 ml per plate and declined at a dose of 8 ml per plate. By this study a significant higher mutagenic potential for hemp biochar compared to wood biochar and tar could be observed suggesting careful application in soil melioration. & 2014 Elsevier Inc. All rights reserved.
Keywords: Biochar Mutagenic potential Pyrolysis Polycyclic aromatic hydrocarbons Ames test
1. Introduction Biochar is a solid material obtained from the hydrothermal carbonisation and pyrolysis of biomass. Biochar application is of gaining interest in the research world. Biochar application in agricultural filed is assumed to increase soil fertility and agricultural productivity, mitigate climate change via carbon and methane sequestration (Sharon and Spokas, 2011). In spite of the advantages of biochar, there are also certain threats related with its production and subsequent utilization. Among these threats, most frequently mentioned are the pollutants of biochar with PAHs introduced during the production process. For example, wood gasifier biochar is known to frequently carry higher amounts of PAHs, and PAH-rich tar is the byproduct of wood biochar (Freddo et al., 2012; Hilber et al., 2012; Keiluweit et al., 2012; Oleszczuk et al., 2013, 2014). Many of these PAHs are recognized as priority pollutants, and their dissemination is heavily regulated by,
n
Corresponding author. E-mail address: [email protected] (R. Anjum).
http://dx.doi.org/10.1016/j.ecoenv.2014.06.005 0147-6513/& 2014 Elsevier Inc. All rights reserved.
as example, the United States Environmental Protection Agency (US EPA) due to their carcinogenic, mutagenic or teratogenic properties (Keiluweit et al., 2012). PAHs can be broadly separated into three nonexclusive categories based on their source: biogenic, petrogenic, and pyrogenic PAHs. Biogenic PAHs are formed from natural biological processes including diagenesis; petrogenic PAHs are derived from petroleum and usually enter the aquatic environment dissolved in water, air, or a cosolvent such as crude oil; and pyrogenic PAHs are formed as a result of incomplete combustion of fuels and largely enter the environment tightly (Thorsen et al., 2004). Chemistry of pyrolysis involves not only the decomposition of organic compounds, but also the formation of highly condensed aromatic structures, organic compounds have two aspects; pyrolysis can destroy compounds present in the feedstock or produce them in the process. This is greatly influenced by the process and its conditions (Bridle et al., 1990; Libra et al., 2011). Hence the production conditions of biochars may encourage the formation of mutagenic substances such as PAH, polychlorinated dibenzodioxins and furans such as polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzofurans (PCDF), phenols and phenol-derivates, or
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further yet unknown toxic chemicals from biogenic material. Also 5-hydroxymethylfurfural (HMF), a harmful phenolic compound can be formed during biochar processes (Busch et al., 2013). 5hydroxymethylfurfural (HMF) is proposed as a crucial intermediate product produced during hydrothermal carbonization and research interest in the production of HMF has a long history (Libra et al., 2011). On the other hand, heavy metals cannot be destroyed during pyrolysis, in contrast to organic compounds. Hence they may have a toxic risk potential (Libra et al., 2011). There are two main potential origins of PAHs in biochar, i.e., the composition of the feedstock and the chemical modification of feedstock compounds by the pyrolysis process. Depending on the feedstock composition and production conditions, varying concentrations of PAHs, polychlorinated biphenyls (PCB) and dioxins can be released during the sorption process of biochar (Smernik, 2009; Singh et al., 2010; Hale et al., 2012). Some of these compounds will be converted during the pyrolysis process and some will remain unchanged or give rise to potentially harmful substances (Lehmann et al., 2009; Hammond et al., 2013). Major chemical pathways for PAH formation during pyrolysis process are high temperature based secondary and tertiary pyrolysis reactions. The evolution of PAHs from the solid substrate has been reported in the temperature range of 400–600 1C (Hwang et al., 2008; Busch et al., 2013; Oleszczuk et al., 2014). This pathway yields predominantly lower molecular weight PAHs, although higher molecular weight PAHs, such as benzo(a)pyrene, are also formed (McGrath et al., 2001, 2003, 2007; Freddo et al., 2012; Busch et al., 2013). The physical form of pyrolysis products may present a direct health risk, or increase or decrease the risk of elements, compounds or crystalline material both in feedstock or formed during pyrolysis (Ryu et al., 2007; Lehmanns et al., 2009). Consequently, before agricultural soil application, it was a necessity to test hydrochars and biochars for their potential genotoxicity (Busch et al., 2012). Over the last decades, more than 200 short term bioassays utilizing micro-organisms, insects or plants have been developed and used to help identify agents that pose genotoxic hazards. Among these tests, mutagenicity bioassays have been proved to be a very useful environmental monitoring and assessment of pollution (Ansari and Malik, 2008). These bioassays provide a means for assessing the genotoxicity of complex mixtures without the need of precise chemical characterization (Chenon et al., 2003; Martin et al., 2005; Mouchet et al., 2006; Lah et al., 2008). The Salmonella mutagenicity test for mutagens and environmental compounds is the most widely used short-term test (Ames et al., 1975; Maron and Ames, 1983; Ansari and Malik, 2008; Anjum and Malik, 2012). The aim of this study is the production and characterization of pyrolysis biochars. Ames Salmonella/mammalian-microsomal mutagenicity test (Maron and Ames, 1983) has been conducted for the assessment of mutagenic potential of native samples of hemp biochar, wood biochar and tar as typical byproduct of wood biochar production to reveal its mutagenicity due to PAHs and phenolic compounds.
2. Materials and methods 2.1. Materials used in pyrolysis for biochar production For the pyrolysis process two different types of biomasses were used. One was premium hemp bedding used as herbaceous feedstock for horses and ponies (DUN Argo-Oude Pekela, Netherlands). Prior to pyrolysis, the particle size of hemp was around 1 cm. The second feedstock was commercial wood pellets (Flammence Holzpellet, Germany) purchased from local market (Potsdam, Germany). The cylindric shaped wood pellets were approximately 2–5 cm long with diameter of around 6 mm.
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2.2. Biochar characterization For the biochar production a horizontal rotating tube lab scale pyrolyzer was used (HTM Reetz, Germany) with an inner diameter of 13 cm and a length of 1.2 m. A feeder was installed to guarantee a continuous feeding to the pyrolyzer. During the pyrolysis the reactor chamber was heated and rotated simultaneously. The pyrolyzer consists of two air draft cooling sections, an electrically heated furnace, a gas cooling section and five thermocouples. The reactor chamber is located inside the electrically heated furnace and a motor is used to rotate the reactor chamber (120 rpm). Both biomasses were dried at 105 1C for at least 24 h prior to pyrolysis. In the conducted experiments, dry biomass was loaded to the feeder, and the gas condensation system was set up. The reactor system was then purged with nitrogen to avoid combustion. After 5 min of purging, the heater was turned on, and the rotation was started (100–120 rpm). When the reactor reached the desired temperature (500 1C7 20 1C), the feeder motors were turned on and biomass of all samples were introduced to the reactor. The retention time for the sample was 30 75 min. After the retention time, the rotating tube was declined at 31 (the horizontal tube was angled at 31) to ease the biochar movement toward the collection vessel. Biochar was collected in the collection vessel, while the gas was condensed in the water bath. Hemp biochar was made in two different scenarios, one with and the other without gas condensation system. During pyrolysis of hemp, only very small amounts of condensate i.e. tar was produced. In a second experiment wood pellets were pyrolysed applying the same experimental conditions. In this case, major volumes of tar were produced and used for this study. Moisture contents in hemp biochar and wood biochar were determined by oven-drying (24 h at 100 1C). pH of biochars, total C, H, N contents, and cation exchange capacities (CECs) were determined according to Chapman (1965). Total metal concentrations were measured using ICP-OES (inductively coupled plasma atomic emission spectroscopy) after ashing at 750 1C followed by extraction with aqua regia (nitro-hydrochloric acid).
2.3. Chemical analysis of pyrolysis products The analysis of the pyrolysis biochars and tar for polycyclic aromatic PAHs as well as phenolic compounds was carried out according to DIN (German Industry Norm) ISO 18287 (German Institute for Standardization, (2006): Determination of the polycyclic aromatic hydrocarbons (PAH) – Gas chromatographic method with mass spectrometric detection (GC–MS) (ISO 18287:2006)), Eurofins Umwelt, Jena, Germany.
2.4. Extraction of PAHs from pyrolysis products The soxhlet extraction by Eurofins according to DIN ISO 18287 was adapted in this study using toluene as solvent to extract PAHs and phenolic compounds from pyrolysed biochars. For solid biochar, 10 g of biochar was poured into 150 ml of toluene and mixed for 7.5 h. The extracts were evaporated until the last drop of toluene was observed (usually takes 20–30 min) by using a rotatory evaporator (63 Heidolph, Germany). The residues left on the rotary evaporator were then poured into 10 ml acetonitrile. The obtained extracts were then filtered using a syringe filter (PTFE type, pore diameter 0.25 mm). The extracted solution was stored at 20 1C until further study. For tar sample, 10 ml of tar was diluted with acetonitrile, evaporated, filtered, and stored similarly as described above. Pyrolysis wood, biochar extract and tar were diluted 1:10 whereas pyrolysis hemp biochar was diluted 1:15 with acetonitrile before genotoxicity testing was conducted.
2.5. Characterstics of selected Ames strains The Salmonella typhimurium strains were maintained in frozen stocks ( 80 1C, DMSO culture) and grown as described by Maron and Ames (1983). Each strain was tested on the basis of associated genetic markers, raised from a single colony of the master plate (Maron and Ames, 1983). A set of five (TA97, TA98, TA100, TA102, and TA104) Ames strains was used; the characteristics of Salmonella typhimurium are given in Table S1 (Supplemental material). TA97, TA98, and TA100 cause mutation at G–C site whereas TA102 and TA104 causes mutation at A–T site. These sets of Ames strains were selected for this study due to their specific mode of action at G–C and A–T base pair at the critical sites of the reversion in the presence of different chemicals (carcinogens). TA98 and T100 strains are particularly sensitive to the mutagenicity of aromatic amines and polycyclic hydrocarbons. All strains are amino acid-dependent strains of Salmonella typhimurium each carrying different mutations in various genes in the histidine operon. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. In the absence of an external histidine source, cells cannot grow and form colonies. Only those bacteria that revert to histidine independence (his þ ) are able to form colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen (MMS as a positive control and tested biochar extract) was added to the plate, the number of revertant
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colonies per plate was increased, usually in a dose-related manner. Many mutagens and carcinogens found in the environment required metabolic activation to increase the sensitivity of the bacteria to the mutagenic effect of these compounds. Hence metabolic activation S9 was used in parallel with all test experiments. 2.6. Preparation of rat liver microsomal fraction Rat liver microsomal fraction (S9) was prepared according to the method of Garner et al. (1972).
lower than o2.1 and o 0.2 mg N g 1 biochar, respectively. In the field soil adsorption experiment, hemp biochar adsorbed 10.9 7 2.8 mg NH4þ –N g 1 biochar and 12.5 7 4.0 mg NO3 N g 1 biochar, whereas wood biochar adsorbed 4.8 72.1 mg NH4þ –N g 1 biochar and 3.8 71.6 mg NO3 –N g 1 biochar respectively (mean of three replicates). Total Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, W, and Zn toxic and non-esssential heavy metals concentrations were higher in hemp biochar as compare to wood biochar as shown in Table 1.
2.7. Salmonella mutagenicity testing The pre-incubation test was performed as described by Maron and Ames (1983) with minor modifications (Pagano and Zeiger, 1992). Five doses of hemp biochar extract (0.5, 1.0, 1.5, 2.0, and 2.5 ml per plate) and five doses of each wood biochar extract and tar (0.5, 1.0, 2.0, 4.0, and 8.0 ml per plate) were plated in triplicates with 0.1 ml of bacterial culture each. When the test sample and bacterial culture were incubated (30 min at 37 1C), 2.0 ml top agar containing traces of histidine and biotin were added. Contents were poured on minimal glucose agar plates. Plates were incubated at 37 1C for 48–72 h. Negative and positive controls were included in each assay. Negative control plates contained bacteria and acetonitrile solvent but no test sample. Methyl methane sulfonate and sodium azide (Maron and Ames, 1983; Anjum and Malik, 2013) served as positive controls. All extracts were also tested in the absence ( S9) or presence ( þS9) of rat liver microsomal fraction, to which 20 ml of S9 liver homogenate mix per plate was added. The criterion used to classify the results as positive was considered according to the studies of Vargas et al. (1995). 2.8. Statistical analysis Statistical analysis of the results of mutagenicity testing for the different parameters; mutagenic index (MI), mutagenic potential (M), induction factor (I) and analysis of variance (ANOVA) were carried out according to Anjum and Malik (2013). 2.8.1. Mutagenic index (MI) The number of his þ revertants in the sample was compared to the negative control by its mutagenic index value. Mutagenic index ¼
Number of his þ revertants induced in the samples Number of his þ revertants induced in the negative control
2.8.2. Mutagenic potential (M) The mutagenic potential of the test samples was calculated by the initial linear portion of the dose response curve with tester strains. The slope (m) was obtained by the least squares regression of the initial linear portion of the curve of initial dose response. 2.8.3. Induction factor (I) The induction factor for various test strains for different soil extracts was evaluated as follows: n c I ¼ ln c where n is the number of revertants in the samples and c is the number of revertants in the solvent control. The induction factor was calculated to determine the difference between two samples if the sensitivity pattern based on the slope (m). 2.8.4. Analysis of variance (ANOVA) To determine the significance of the number of his þ revertants in the samples compared to the control, one way ANOVA was done at Pr 0.05 using MINITAB Software, USA.
3. Results 3.1. Biochar characterization The total organic carbon of hemp biochar and wood biochar was 92.6% and 84.2% (mean of three replicates) and pH-KCl was 7.1 70.33 and 8.0 70.53 respectively. Nitrogen contents in biochar samples were lower than 4% resulting in high carbon and nitrogen ratio (4 478). The percentage of ash content in hemp biochar (13.1 70.1) was higher than wood biochar (4.2 70.2). NH4þ and NO3 concentrations in hemp biochar and wood biochar were
3.2. Determination of PAHs and phenolic compounds in pyrolysis hemp biochar, wood biochar and tar Gas chromatography and mass spectrometry analysis of biochars and tar samples revealed the presence of various PAHs. The total concentrations of PAHs in hemp biochar and wood biochar were 34.9 mg g 1 of dry mass and 33.7 mg g 1 of dry mass respectively as shown in Table 2; the concentration of PAH in the tar was considerably lower (17.4 mg g 1). Total phenolic compounds of 55 mg g 1 and 8.3 mg g 1 were also quantified in hemp and wood biochar, respectively. No phenolic compounds were detected in the tar sample. The analysis of hemp biochar revealed the presence of petrogenic PAHs i.e. napththaline, acetonaphthyene, acenaphthene and fluorine, whereas the concentrations of petrogenic PAHs (napththaline, acetonaphthyene, acenaphthene, and fluorine) in wood biochar were lower as compared to hemp biochar (Table 2). Pyrogenic PAHs, i.e. phenanthrene, anthracene, fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, and indeno (1,2,3-cd) pyrene were also determined in hemp biochar, however wood biochar revealed the presence of pyrogenic PAHs at a higher concentration as compared to hemp biochar (Table 2). It was observed that the concentration of pyrogenic and petrogenic PAHs in tar was lower than hemp biochar and wood biochar.
Table 1 Quantitative determination of toxic and non-esssential heavy metals in hemp biochar and wood biochar by ICP-OES analysis. Metals in sample
Al As B Ca Cd Co Cr Cu Fe K Mg Mn Mo Na Ni P Pb S Se W Zn
Concentration in dry biomass [mg g 1] Hemp biochar
Wood biochar
761 o 0.01 36.2 81,911 o 0.01 1.67 o 0.01 66.7 92.1 18,819 5705 565 o 0.01 2444 5.86 17,845 0.74 4768 1.56 o 0.01 528
72.4 o 0.01 1.58 1156 o 0.01 0.08 0.38 2.5 273 2944 428 23.7 0.15 326 0.28 763 0.35 362 0.07 o 0.01 12.2
Each value is the mean of three duplicates.
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Table 2 Quantitative determination of polycyclic aromatic hydrocarbons (PAHs) in pyrolysis biochars and tar by gas chromatography and mass spectrometry. Concentration in dry biomass [mg g 1]
PAHs (petrogenic and pyrogenic)
Naphthaline Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benz(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Dibenz(a,h)anthracene Benzo(g,h,i)perylene Total
Hemp biochar
Wood biochar
Tar (byproduct)
7.1 3.1 2.0 4.6 9.3 2.9 2.0 2.6 0.5 0.5 0.1 o0.05 0.1 o0.05 o0.05 o0.05 34.9
3.8 2.3 1.2 3.2 11 3.2 2.4 3.2 1.3 1.2 0.3 0.1 0.3 0.06 o 0.05 o 0.05 33.7
5.8 0.7 0.8 1.5 6.0 1.5 0.7 0.3 o0.05 o0.05 o0.05 o0.05 o0.05 o0.05 o0.05 o0.05 17.4
Each value is the mean of three duplicates.
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3.3. Reversion of the Salmonella tester strains in the presence of biochar and tar extracts Mutagenicity testing of biochars and tar was conducted by Ames tests. Five doses of hemp biochar extract (0.5, 1.0, 1.5, 2.0, and 2.5 ml per plate) and five doses of each wood biochar extract and tar (0.5, 1.0, 2.0, 4.0, and 8.0 ml per plate) were plated in triplicates with 0.1 ml of bacterial culture each. Positive and negative control of each experiment was run in parallel at the same five doses. Positive control (methyl methane sulfonate and sodium azide), biochars and tar extracts revealed reversion of Ames strains, whereas with negative control (acetonitrile solvent) at the same dose reversion in tester strains were not observed. Consequently, negative control test indicated that acetonitrile solvent was not mutagenic. The number of reverted tester strains are shown in Figs. 1–3 and reversion results (in terms of MI, I, m and LSD) of Salmonella typhimurium strains with hemp biochar, wood biochar and tar are summarized in Table 3. The data interpretation by statistical analysis in terms of mutagenic index, mutagenic potential, induction factor and analysis of variance indicates that biochar and tar samples exhibited maximum mutagenicity with TA98 and TA100 strains in the presence and absence of rat liver microsomal fraction (S9).
Table 3 Reversion of Salmonella tester strains in the presence of hemp biochar, wood biochar and tar in terms of mutagenic index (MI), induction factor (I), slope (m) of the initial linear dose response curve as determined by linear regression analysis and least significance difference (LSD). Salmonella strains
TA97 TA98 TA100 TA102 TA104 Salmonella strains
S9
Hemp biochar: mutagenic index (MI) 0.5 ml per plate
1.0 ml per plate
1.5 ml per plate
2.0 ml per plate
2.5 ml per plate
þ þ þ þ þ
2.7 2.7 8.5 8.3 2.0 2.3 1.1 1.6 1.5 1.5
4.0 3.9 10.0 12.0 2.7 3.3 1.3 2.0 1.6 1.8
4.5 4.7 14.8 15.5 3.1 4.0 2.1 2.2 1.8 2.0
5.0 5.4 15.3 17.0 3.5 4.3 2.3 2.7 2.1 2.2
3.7 4.3 12.2 13.3 2.7 3.6 1.9 2.1 1.7 1.7
S9
Wood biochar: mutagenic index (MI) 0.5 ml per plate
TA97 TA98 TA100 TA102 TA104 Salmonella strains
þ þ þ þ þ
1.1 1.2 1.8 1.9 1.2 1.4 1.1 1.2 1.0 1.0
S9
Tar: mutagenic index (MI) 0.5 ml per plate
TA97 TA98 TA100 TA102 TA104
1.0 ml per plate
þ þ þ þ þ
1.2 1.2 1.7 2.2 1.3 1.4 1.2 1.3 1.1 1.1
1.8 1.7 3.3 3.1 1.7 1.8 1.3 1.4 1.2 1.2
1.0 ml per plate 1.7 1.5 2.7 2.8 1.6 1.6 1.3 1.4 1.2 1.2
2.0 ml per plate 2.0 2.0 4.2 3.9 2.1 2.2 1.4 1.5 1.3 1.3
4.0 ml per plate 2.4 2.4 5.4 4.9 2.7 2.6 1.6 1.7 1.4 1.4
I
m
LSD P r 0.05
1.39 1.49 2.66 2.77 0.93 1.20 0.26 0.54 0.10 0.14
14.7 18.9 21.2 25.2 16.2 20.1 14.6 17.3 16.2 15.8
8.9 4.1 5.2 21.4 4.1 3.1 2.3 20.4 12.2 30.3
I
m
LSD P r 0.05
8.0 ml per plate 2.3 2.2 5.0 4.2 2.4 2.3 1.5 1.6 1.2 1.2
0.30 0.35 1.49 1.35 0.52 0.46 0.50 0.32 1.01 1.03 I
2.0 ml per plate 2.1 1.8 3.0 3.2 1.8 1.9 1.4 1.5 1.2 1.3
4.0 ml per plate 2.2 2.1 3.4 3.9 2.8 2.9 1.5 1.6 1.3 1.4
Abbreviations: S9, liver microsomal fraction; , in the absence of S9; þ , in the presence of S9.
3.5 3.7 3.9 3.8 5.7 5.6 3.3 4.4 2.9 2.8 m
9.7 8.8 4.8 4.8 2.5 5.5 2.9 12.4 3.6 8.3 LSD P r 0.05
8.0 ml per plate 1.9 1.8 2.8 3.1 2.0 2.0 1.1 1.2 1.0 1.1
0.25 0.13 0.89 1.09 0.36 0.37 0.60 0.40 1.16 0.97
2.7 2.9 1.8 2.7 4.9 4.3 2.6 3.3 2.5 3.1
17.5 12.2 4.5 12.7 5.0 3.2 2.8 14.4 4.3 5.7
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Fig. 1. Hemp biochar: number of reverted tester strains TA97, TA98, TA100, TA102, and TA104 in the absence ( S) or in the presence ( þS) of liver microsomal fraction on increased dose (0.5, 1.0, 1.5, 2.0 and 2.5 ml per plate) of hemp biochar. Control¼ without extract. * ¼ indicates maximum number of reverted tester strains.
Fig. 2. Wood biochar: number of reverted tester strains TA97, TA98, TA100, TA102, and TA104 in the absence ( S) or in the presence ( þS) of liver microsomal fraction on increased dose (0.5, 1.0, 2, 4.0 and 8.0 ml per plate) of wood biochar. Control¼ without extract. * ¼ indicates maximum number of reverted tester strains.
The number of reversion of tester strains increased in both presence and absence of S9 with an increasing dose of hemp biochar extract up to 2 ml per plate whereas a decline was observed at concentrations of 2.5 ml per plate (Fig. 1). Consequently, hemp biochar sample exhibit maximum mutagenicity with TA97, TA98 and T100 strains both in the presence and absence of S9 fraction. An increasing dose of test samples caused a gain of reverted tester strains (Fig. 1). While for wood biochar and tar extract a reversion of tester strains increased in the presence and absence of S9 with concentrations of extract up to 4 ml per plate whereas reversion was sharply decline at the dose of 8 ml per plate. Wood biochar and tar extract exhibited maximum mutagenicity with TA98 and TA100 strains in the presence and absence of rat liver (S9) microsomal fraction (Figs. 2 and 3). According to mutagenic index TA98 and TA100 was the most responsive strain against all test samples while other strains revealed weak response in comparison to the TA98 strain
Fig. 3. Tar: number of reverted tester strains TA97, TA98, TA100, TA102, and TA104 in the absence ( S) or in the presence ( þS) of liver microsomal fraction with increased dose (0.5, 1.0, 2, 4.0 and 8.0 ml per plate).
(Table 3). Highest mutagenic index value was observed for hemp biochar extract. Consequently, in terms of mutagenic index hemp biochar was the most mutagenic than the wood biochar and tar. The response of tester strains in terms of slope (m) of the initial linear dose response curve was obtained by the least squares regression. It was found that TA98 and TA100 showed the maximum slope value in hemp biochar extract. Only TA100 showed the maximum slope value in wood biochar and tar extract (Table 3). According to the observed value of induction factor (I), TA98 was the most responsive strain against all test samples while other strains revealed weak response in comparison to the TA98 strain (Table 3). However, biochar extracts and tar exhibited a variable trend in responsiveness of the different strains in the presence of S9 fraction in terms of slope with respect to the initial dose response curve. The significance of the reversion of tester strains with increasing doses was determined by one way ANOVA. The mutagenic potential of the hemp biochar, wood biochar, and tar extract was calculated by the initial linear portion of the dose response curve with tester strains. The mutagenicity for hemp biochar extracts in terms of mutagenic potential (M) of the initial linear dose response for the most responsive strain TA98 with and without S9 was higher as compared to wood biochar and tar. Net revertants per gram of hemp biochar, wood biochar and tar were 4,485,000, 522,000 and 387,500, respectively, for most responsive TA98 strains in the presence of S9 fraction. The maximum number of net revertants per gram of hemp biochar, wood biochar and tar in the absence of S9 fraction were 3,690,000, 435,000 and 310,000, respectively, for most responsive TA98 strains. The mutagenic activity was slightly enhanced in the presence of S9 for both biochar and tar extracts tested.
4. Discussion For the study of mutagenicity assessment of pyrolysis biochars, hemp biochar and wood biochar were produced by different types of feed stocks with same biochar production processes. The characteristics of hemp biochar and wood biochars were variably different from each other. We have been adopted Ames assay for the evaluation of risks associated with the handling and use of
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biochars. The result of this study reveals mutagenic potential of pyrolysis biochars. In our study we observed and discussed different mutagenic responses against biochars that are comparable to each other and also focused on the by-product tar. In the present study, characterized hemp biochar revealed higher adsorption of NH4þ –N g 1 biochar and NO3 –N g 1 biochar in the field soil as compared to wood biochar. On the other hand, ICP-OES analysis of hemp biochar and wood biochar for toxic and non-essential heavy metals revealed low concentrations of toxic heavy metals (As, Cr, Cd i.e. o0.01) as shown in Table 1, those may not have any toxic or mutagenic effect. In our study we have determined a combinations of petrogenic and pyrogenic PAHs i.e. napththaline, acetonaphthyene, acenaphthene, and fluorine; these were higher in hemp biochar as compared to wood biochar and tar. The concentration of pyrogenic PAHs i.e. phenanthrene, anthracene, fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, and indeno(1,2,3cd) pyrene were higher in wood biochar than hemp biochar. Here data of chemical analysis of biochars revealed the presence of greater abundance of pyrogenic PAHs as shown is Table 2. Comparable to wood biochar and tar, high mutagenicity was observed due to the presence of high concentration of petrogenic PAHs in hemp biochar. Hence, the results of this study indicate that petrogenic PAHs were more mutagenic than pyrogenic PAHs. Oleszczuk et al. (2014) collected soil samples in the vicinity of biochar production sites and detected polycyclic aromatic hydrocarbons (PAH). In their study, the concentrations of PAHs were 1796–101,282 μg kg 1, much higher than our study, however, temperature (400–500 1C) inside the kiln was same as in our study. Fabbri et al. (2013) also analyzed PAHs in biochar and biochar amended soil produced by slow pyrolysis from woody biomass, whereas we applied fast pyrolysis. Fabbri et al. (2013) reported a total PAH levels ranged between 1.2–19 μg g 1 and 0.2–5.0 μg g 1. Specifically, benzo(a)pyrene ranging between 0.01 and 0.67 μg g 1 across various biochars amended soils which were much higher than our biochar samples. Therefore, our data and other studies (Fabbri et al., 2013; Oleszczuk et al., 2014) indicate that some factors were influencing the production of mutagenic substances which was slow and fast pyrolysis process and temperature inside the kiln might be also a cause of production of mutagens (PAH). It can be assumed that pollutants of biochar might be overcome due to the further optimization of pyrolysis process. In addition, our results also suggest optimizing the pyrolysis to the type of biomass feedstock. The concentration of pyrogenic PAH was noticed much higher in hemp biochar as compared to wood biochar; and in mutagenicity testing, higher mutagenic potential of hemp biochar was observed as compared to wood biochar. Ames test by Maron and Ames (1983) is one of the important assays for testing of environmental samples. in vitro Salmonella/ microsomal assay with rat liver microsomal fraction (þS9) is also one of the most frequently used test for assessing the mutagenic potential for both pure compounds and complex mixture (Ames et al., 1975). An evidence of earlier study by Ames et al. (1975) revealed that fluorene and benzo(a)pyrene is a mutagen which has been detected in biochar samples (Table 2). In their study, 2-aminofluorene at the concentration of 2 mg per plate was mutagenic, and it acts as a direct mutagen which caused frame shift mutation at G–C site of TA98 and TA100 tester strains. Similar to Ames et al. (1975), in our study TA98 and TA100 tester strains were the most responsive tester strains. The concentrations of fluorine in hemp biochar and wood biochar were 4.6 mg g 1 and 3.2 mg g 1 respectively, whereas, concentrations of benzo(a)pyrene in hemp biochar and wood biochar were 0.18 mg g 1 and 0.37 mg g 1, respectively (Table 2). Maximum mutagenicity was observed in hemp biochar extract at
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the highest concentration of 2.0 ml per plate and observed 492 revertants in the absence of S9 and 598 revertants in the presence of S9 for most responsive TA98 strain can be observed in Fig. 1. Ames et al. (1975) also reported, when TA100 was treated with benzo(a)pyrene at concentration of 5 ml per plate and in the presence of S9 at concentration of 20 ml per plate, they observed 318 revertants per plate which is comparable to our study as 358 and 328 revertants per plate were obtained when TA100 was treated with wood biochar and tar (Figs. 2 and 3). Consequently our data indicates that pyrolysis biochars caused frame shift mutation at G–C site of TA98 and TA100 tester strains which revealed mutagenicity response due to detected PAHs in hemp biochar, wood biochar, and tar. Beside Ames Salmonella/microsomal mutagenicity test, genotoxicity assessment of biochar has employed many assays to detect DNA damage ability and mutagenicity such as Tradescantia micronucleus test and Microtoxs toxicity test ( Busch et al., 2013; Oleszczuk et al., 2014), and results of their studies are discussed here. Oleszczuk et al. (2014) performed several ecotoxicological tests (phytotoxicity to Lepidiumsativum, Microtoxs-Vibrio fischeri, MARA-11 different microorganisms, Daphtoxkit F™-Daphnia magna) to evaluate the effect of polluted soil by PAHs on plant, bacteria and crustaceans. Depending on the ecotoxicological assay applied and sampling sites, the toxicity of the soils was varied, but only in a few cases the phytotoxicity was correlated with the content of PAHs. Busch et al. (2013) tested genotoxic effect of different hydrochars and biochars with the Tradescantia micronucleus test and observed chromosomal aberrations in pollen cells of Tradescantia. These findings as above resulting from a completely different approach support our findings and underline the necessity of exhaustive risk assessment of biochars, namely of their processing and application.
5. Conclusion Only a few reports exist describing genotoxic impacts of biochar. As biochar production linked to mutagenicity and toxicity is a relatively new issue, this work provides first and new insights to this topic. In Ames Salmonella/microsomal mutagenicity test system, it was observed, that pyrolysis hemp biochar exhibited higher mutagenic effects than wood biochar and tar. As example, the Ames method clearly indicates that petrogenic PAHs and pyrogenic PAHs within the biochar caused frame shift mutation at G–C site of TA98 and TA100 tester strains. Consequently, our finding demands caution in handling of biochars, since the unknown mutagens may also impact on human health. Further research is urgently needed to optimize the biochar production process and to identify the unknown mutagens in biochars including their impact on biological systems.
Acknowledgments R.A. gratefully acknowledges the fellowship grant provided by the Leibniz Association and German Academic Exchange Service (DAAD, Grant no. A/12/93888). M.T.R. and N.K. are thankful for grants provided by the German Federal Ministry of Research and Education (BMBF, Grant no. 03SF0381A) and the German Federal Ministry of Food and Agriculture (BMEL, Grant no. 22026411). The authors thank T. Nohmi, National Institute of Hygienic Sciences, Division of Genetics and Mutagenesis, Tokyo, Japan, for the generous provision of Ames tester strains. Additionally, the authors would like to thank K. Mundt, T. Langer, U. Lüder and G. Franke for experimental support and C. Prautsch and L. Herkoltz for their support in analytical tasks. The author's sincere thanks
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also go to the three anonymous reviewers who provided critical constructive comments on the manuscript, which resulted in a significantly improved article. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.06.005. References Ames, B.N., Kammen, H.O., Yamasaki, E., Lee, F.D., 1975. Methods for detecting carcinogens and mutagens with Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31, 347–364. Anjum, R., Malik, A., 2012. Mutagenicity assessment of contaminated soil in the vicinity of industrial area. Environ. Monit. Assess. 184, 3013–3026. Anjum, R., Malik, A., 2013. Evaluation of mutagenicity of wastewater in the vicinity of pesticide industry. Environ. Toxicol. Pharmacol. 35, 285–291. Ansari, M.I., Malik, A., 2008. Genotoxicity of wastewaters used for irrigation of food crops. Environ. Toxicol. 24, 103–115. Bridle, T., Hammerton, I., Hertle, C., 1990. Control of heavy-metals and organochlorines using the oil from sludge process. Water Sci. Technol. 22, 249–258. Busch, D., Kammann, C., Grünhage, L., Müller, C., 2012. Simple biotoxicity tests for evaluation of carbonaceous soil additives: establishment and reproducibility of four test procedures. J. Environ. Qual. 4, 1023–3241. Busch, D., Stark, A., Kammann, C.I., Glaser, B., 2013. Genotoxic and phytotoxic risk assessment of fresh and treated hydrochar from hydrothermal carbonization compared to biochar from pyrolysis. Ecotoxicol. Environ. Saf. 97, 59–66. Chapman, H.D., 1965. Cation-exchange capacity. In: Black, C.A. (Ed.), Methods of Soil Analysis: Part 2 – Chemical and Microbiological Properties. American Society of Agronomy, Madison, WI, USA, pp. 891–901. Chenon, P., Gauthier, L., Loubieres, P., Severac, A., Delpoux, M., 2003. Evaluation of the genotoxic and teratogenic potential of a municipal sludge and sludge amended soil using the amphibian Xenopus laevis and the tobacco: Nicotiana tabacum L. var. xanthi dulieu. Sci. Total Environ. 301, 139–150. Fabbri, D., Rombolà, A.G., Torri, C., Spokas, K.A., 2013. Determination of polycyclic aromatic hydrocarbons in biochar and biochar amended soil. J. Anal. Appl. Pyrolysis 103, 60–67. Freddo, A., Cai, C., Reid, B.J., 2012. Environmental contextualisation of potential toxic elements and polycyclic aromatic hydrocarbons in biochar. Environ. Pollut. 171, 18–24. Garner, R.C., Miller, E.C., Miller, J.A., 1972. Liver microsomal metabolism of aflatoxin B1 to a reactive derivative toxic to Salmonella typhimurium TA1530. Cancer Res. 32, 2058–2066. Hale, S.E., Lehmann, J., Rutherford, D., Zimmerman, A.R., Bachmann, R.T., Shitumbanuma, V., O’Toole, A., Sundqvist, K.L., Arp, H.P.H., Cornelissen, G., 2012. Quantifying the total and bioavailable polycyclic aromatic hydrocarbons and dioxins in biochars. Environ. Sci. Technol. 46, 2830–2838. Hammond, J., Shackley, S., Prendergast-Miller, M., Cook, J., Buckingham, S., Pappa, V., 2013. Biochar field testing in the UK: outcomes and implications for use. Carbon Manag. 4, 159–170. Hilber, I., Blum, F., Leifeld, J., Schmidt, H.-P., Bucheli, T.D., 2012. Quantitative determination of PAHs in biochar: a prerequisite to ensure its quality and safe application. J. Agric. Food Chem. 60, 3042–3050. Hwang, I.H., Nakajima, D., Matsuto, T., Sugimoto, T., 2008. Improving the quality of waste-derived char by removing ash. Waste Manag. 28, 424–434.
Keiluweit, M., Kleber, M., Sparrow, M.A., Simoneit, B.R.T., Prahl, F.G., 2012. Solvent extractable polycyclic aromatic hydrocarbons in biochar: influence of pyrolysis temperature and feedstock. Environ. Sci. Technol. 46, 9333–9341. Lah, B., Vidic, T., Glasencnik, E., Cepeljnik, T., Gorjanc, G., Marinsek-Logar, R., 2008. Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay. Environ. Monit. Assess. 139, 107–118. Lehmann, J., Czimczic, C., Laird, D., Sohi, S., 2009. Stability of biochar in soil. In: Lehmann, J., Joseph, S. (Eds.), Biochar for Environmental Management. Earthscan, London, pp. 317–340. Libra, J.A., Ro, K.S., Kammann, C., Funke, A., Berge, N.D., Neubauer, Y., Titirici, M.M., Fühner, C., Bens, O., Kern, J., Emmerich, K.H., 2011. Hydrothermal carbonization of biomass residuals: a comparative review of the chemistry, processes and applications of wet and dry pyrolysis. Biofuels 2, 71–106. Maron, D.M., Ames, B.N., 1983. Revised methods for Salmonella mutagenicity test. Mutat. Res. 101, 173–215. Martin, F.L., Piearce, T.G., Hewer, A., Phillips, D.H., Semple, K.T., 2005. A biomarker model of sublethal genotoxicity (DNA single-strand breaks and adducts) using the sentinel organism Aporrectodea longa in spiked soil. Environ. Pollut. 138, 307–315. McGrath, T., Sharma, R., Hajaligol, M., 2001. An experimental investigation into the formation of polycyclic-aromatic hydrocarbons (PAH) from pyrolysis of biomass materials. Fuel 80, 1787–1797. McGrath, T.E., Chan, W.G., Hajaligol, M.R., 2003. Low temperature mechanism for the formation of polycyclic aromatic hydrocarbons from the pyrolysis of cellulose. J. Anal. Appl. Pyrolysis 66, 51–70. McGrath, T.E., Wooten, J.B., Geoffrey Chan, W., Hajaligol, M.R., 2007. Formation of polycyclic aromatic hydrocarbons from tobacco: the link between low temperature residual solid (char) and PAH formation. Food Chem. Toxicol. 45, 1039–1050. Mouchet, F., Gauthier, L., Mailhes, C., Jourdain, M.J., Ferrier, V., Triffault, G., 2006. Biomonitoring of the genotoxic potential of aqueous extracts of soils and bottom ash resulting from municipal solid waste incineration using the comet and micronucleus tests on amphibian (Xenopus laevis) larve and bacterial assays (Mutatoxs and Ames tests). Sci. Total Environ. 355, 232–246. Oleszczuk, P., Jośko, I., Kuśmierz, M., 2013. Biochar properties regarding to contaminants content and ecotoxicological assessment. J. Hazard. Mater. 260, 375–382. Oleszczuk, P., Kuśmierz, M., Futa, B., Wielgosz, E., Ligęza, S., Pranagal, J., 2014. Microbiological, biochemical and ecotoxicological evaluation of soils in the area of biochar production in relation to polycyclic aromatic hydrocarbon content. Geoderma 213, 502–511. Pagano, D.A., Zeiger, E., 1992. Conditions for detecting the mutagenicity of divalent metals in Salmonella typhimurium. Environ. Mol. Mutagen 19, 136–146. Ryu, C., Sharifi, V.N., Swithenbank, J., 2007. Waste pyrolysis and generation of storable char. Int. J. Energy Res. 31, 177–191. Sharon, L.W., Spokas, K.A., 2011. Impact of biochar on earthworm populations: a review. Appl. Environ. Soil Sci. 2011, 1–12. Singh, B.P., Hatton, B.J., Singh, B., Cowie, A.L., Kathuria., A., 2010. Influence of biochars on nitrous oxide emission and nitrogen leaching from two contrasting soils. J. Environ. Qual. 39, 1224–1235. Smernik, R.J., 2009. Biochar and sorption of organic compounds. In: Lehmann, J., Joseph, S. (Eds.), Biochar for Environmental Management: Science and Technology. Earthscan, London, pp. 289–300. Thorsen, W.A., Cope, W.G., Shea, D., 2004. Bioavailability of PAHs: effects of soot carbon and PAH source. Environ. Sci. Technol. 38, 2029–2037. Vargas, V.M.F., Guidobono, R.R., Jordao, C., Henriques, J.A.P., 1995. Use of two shortterm tests to evaluate the genotoxicity of river water treated with different concentration/extraction procedures. Mutat. Res. 343, 31–52.
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Assessment of mutagenic potential of pyrolysis biochars by Ames Salmonella/mammalian-microsomal mutagenicity test Reshma Anjum n, Niclas Krakat, M. Toufiq Reza, Michael Klocke Leibniz Institute for Agricultural Engineering Potsdam-Bornim e.V., Max-Eyth-Allee 100, D-14469 Potsdam, Germany
art ic l e i nf o
a b s t r a c t
Article history: Received 23 March 2014 Received in revised form 3 June 2014 Accepted 4 June 2014 Available online 20 July 2014
Biochar is of raising interest in sustainable biomass utilization concepts. Particularly biochar derived from pyrolysis attaches important agricultural capacities mandatory for an improved carbon sequestration, soil fertility and amelioration, respectively. In fact, large scale field trials and commercial business with biochar materials have already been started but still only few are known about the mutagenic potential of biochars produced. In this study hemp bedding and wood pellet biomass were used for biochar production by pyrolysis. The total concentrations of polycyclic aromatic hydrocarbons (PAHs) were 34.9 mg g 1 of dry mass and 33.7 mg g 1 of dry mass for hemp biochar and wood biochar, respectively. The concentration of PAHs in tar produced during wood carbonization was 17.4 mg g 1. The concentrations of phenolic compounds were 55 mg g 1 and 8.3 mg g 1 for hemp and wood biochar, respectively. Salmonella/microsomal mutagenicity tests (i.e. Ames test) revealed a maximum mutagenicity for hemp biochar extracts with strains TA97, TA98 and TA100 in the presence and absence of liver microsomal fractions, respectively. Wood biochar and tar extract exhibited maximum mutagenicity with strains TA98 and T100 both in the presence and absence of liver microsomal fraction. The reversion of the applied tester strains increased in the presence and absence of liver microsomal fractions with an increasing dose of hemp biochar extract up to 2 ml per plate and decreased at a concentration of 2.5 ml per plate. For wood biochar and tar extracts, reversion of tester strains increased both in the presence and absence of S9 at extract concentrations of 4 ml per plate and declined at a dose of 8 ml per plate. By this study a significant higher mutagenic potential for hemp biochar compared to wood biochar and tar could be observed suggesting careful application in soil melioration. & 2014 Elsevier Inc. All rights reserved.
Keywords: Biochar Mutagenic potential Pyrolysis Polycyclic aromatic hydrocarbons Ames test
1. Introduction Biochar is a solid material obtained from the hydrothermal carbonisation and pyrolysis of biomass. Biochar application is of gaining interest in the research world. Biochar application in agricultural filed is assumed to increase soil fertility and agricultural productivity, mitigate climate change via carbon and methane sequestration (Sharon and Spokas, 2011). In spite of the advantages of biochar, there are also certain threats related with its production and subsequent utilization. Among these threats, most frequently mentioned are the pollutants of biochar with PAHs introduced during the production process. For example, wood gasifier biochar is known to frequently carry higher amounts of PAHs, and PAH-rich tar is the byproduct of wood biochar (Freddo et al., 2012; Hilber et al., 2012; Keiluweit et al., 2012; Oleszczuk et al., 2013, 2014). Many of these PAHs are recognized as priority pollutants, and their dissemination is heavily regulated by,
n
Corresponding author. E-mail address: [email protected] (R. Anjum).
http://dx.doi.org/10.1016/j.ecoenv.2014.06.005 0147-6513/& 2014 Elsevier Inc. All rights reserved.
as example, the United States Environmental Protection Agency (US EPA) due to their carcinogenic, mutagenic or teratogenic properties (Keiluweit et al., 2012). PAHs can be broadly separated into three nonexclusive categories based on their source: biogenic, petrogenic, and pyrogenic PAHs. Biogenic PAHs are formed from natural biological processes including diagenesis; petrogenic PAHs are derived from petroleum and usually enter the aquatic environment dissolved in water, air, or a cosolvent such as crude oil; and pyrogenic PAHs are formed as a result of incomplete combustion of fuels and largely enter the environment tightly (Thorsen et al., 2004). Chemistry of pyrolysis involves not only the decomposition of organic compounds, but also the formation of highly condensed aromatic structures, organic compounds have two aspects; pyrolysis can destroy compounds present in the feedstock or produce them in the process. This is greatly influenced by the process and its conditions (Bridle et al., 1990; Libra et al., 2011). Hence the production conditions of biochars may encourage the formation of mutagenic substances such as PAH, polychlorinated dibenzodioxins and furans such as polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzofurans (PCDF), phenols and phenol-derivates, or
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further yet unknown toxic chemicals from biogenic material. Also 5-hydroxymethylfurfural (HMF), a harmful phenolic compound can be formed during biochar processes (Busch et al., 2013). 5hydroxymethylfurfural (HMF) is proposed as a crucial intermediate product produced during hydrothermal carbonization and research interest in the production of HMF has a long history (Libra et al., 2011). On the other hand, heavy metals cannot be destroyed during pyrolysis, in contrast to organic compounds. Hence they may have a toxic risk potential (Libra et al., 2011). There are two main potential origins of PAHs in biochar, i.e., the composition of the feedstock and the chemical modification of feedstock compounds by the pyrolysis process. Depending on the feedstock composition and production conditions, varying concentrations of PAHs, polychlorinated biphenyls (PCB) and dioxins can be released during the sorption process of biochar (Smernik, 2009; Singh et al., 2010; Hale et al., 2012). Some of these compounds will be converted during the pyrolysis process and some will remain unchanged or give rise to potentially harmful substances (Lehmann et al., 2009; Hammond et al., 2013). Major chemical pathways for PAH formation during pyrolysis process are high temperature based secondary and tertiary pyrolysis reactions. The evolution of PAHs from the solid substrate has been reported in the temperature range of 400–600 1C (Hwang et al., 2008; Busch et al., 2013; Oleszczuk et al., 2014). This pathway yields predominantly lower molecular weight PAHs, although higher molecular weight PAHs, such as benzo(a)pyrene, are also formed (McGrath et al., 2001, 2003, 2007; Freddo et al., 2012; Busch et al., 2013). The physical form of pyrolysis products may present a direct health risk, or increase or decrease the risk of elements, compounds or crystalline material both in feedstock or formed during pyrolysis (Ryu et al., 2007; Lehmanns et al., 2009). Consequently, before agricultural soil application, it was a necessity to test hydrochars and biochars for their potential genotoxicity (Busch et al., 2012). Over the last decades, more than 200 short term bioassays utilizing micro-organisms, insects or plants have been developed and used to help identify agents that pose genotoxic hazards. Among these tests, mutagenicity bioassays have been proved to be a very useful environmental monitoring and assessment of pollution (Ansari and Malik, 2008). These bioassays provide a means for assessing the genotoxicity of complex mixtures without the need of precise chemical characterization (Chenon et al., 2003; Martin et al., 2005; Mouchet et al., 2006; Lah et al., 2008). The Salmonella mutagenicity test for mutagens and environmental compounds is the most widely used short-term test (Ames et al., 1975; Maron and Ames, 1983; Ansari and Malik, 2008; Anjum and Malik, 2012). The aim of this study is the production and characterization of pyrolysis biochars. Ames Salmonella/mammalian-microsomal mutagenicity test (Maron and Ames, 1983) has been conducted for the assessment of mutagenic potential of native samples of hemp biochar, wood biochar and tar as typical byproduct of wood biochar production to reveal its mutagenicity due to PAHs and phenolic compounds.
2. Materials and methods 2.1. Materials used in pyrolysis for biochar production For the pyrolysis process two different types of biomasses were used. One was premium hemp bedding used as herbaceous feedstock for horses and ponies (DUN Argo-Oude Pekela, Netherlands). Prior to pyrolysis, the particle size of hemp was around 1 cm. The second feedstock was commercial wood pellets (Flammence Holzpellet, Germany) purchased from local market (Potsdam, Germany). The cylindric shaped wood pellets were approximately 2–5 cm long with diameter of around 6 mm.
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2.2. Biochar characterization For the biochar production a horizontal rotating tube lab scale pyrolyzer was used (HTM Reetz, Germany) with an inner diameter of 13 cm and a length of 1.2 m. A feeder was installed to guarantee a continuous feeding to the pyrolyzer. During the pyrolysis the reactor chamber was heated and rotated simultaneously. The pyrolyzer consists of two air draft cooling sections, an electrically heated furnace, a gas cooling section and five thermocouples. The reactor chamber is located inside the electrically heated furnace and a motor is used to rotate the reactor chamber (120 rpm). Both biomasses were dried at 105 1C for at least 24 h prior to pyrolysis. In the conducted experiments, dry biomass was loaded to the feeder, and the gas condensation system was set up. The reactor system was then purged with nitrogen to avoid combustion. After 5 min of purging, the heater was turned on, and the rotation was started (100–120 rpm). When the reactor reached the desired temperature (500 1C7 20 1C), the feeder motors were turned on and biomass of all samples were introduced to the reactor. The retention time for the sample was 30 75 min. After the retention time, the rotating tube was declined at 31 (the horizontal tube was angled at 31) to ease the biochar movement toward the collection vessel. Biochar was collected in the collection vessel, while the gas was condensed in the water bath. Hemp biochar was made in two different scenarios, one with and the other without gas condensation system. During pyrolysis of hemp, only very small amounts of condensate i.e. tar was produced. In a second experiment wood pellets were pyrolysed applying the same experimental conditions. In this case, major volumes of tar were produced and used for this study. Moisture contents in hemp biochar and wood biochar were determined by oven-drying (24 h at 100 1C). pH of biochars, total C, H, N contents, and cation exchange capacities (CECs) were determined according to Chapman (1965). Total metal concentrations were measured using ICP-OES (inductively coupled plasma atomic emission spectroscopy) after ashing at 750 1C followed by extraction with aqua regia (nitro-hydrochloric acid).
2.3. Chemical analysis of pyrolysis products The analysis of the pyrolysis biochars and tar for polycyclic aromatic PAHs as well as phenolic compounds was carried out according to DIN (German Industry Norm) ISO 18287 (German Institute for Standardization, (2006): Determination of the polycyclic aromatic hydrocarbons (PAH) – Gas chromatographic method with mass spectrometric detection (GC–MS) (ISO 18287:2006)), Eurofins Umwelt, Jena, Germany.
2.4. Extraction of PAHs from pyrolysis products The soxhlet extraction by Eurofins according to DIN ISO 18287 was adapted in this study using toluene as solvent to extract PAHs and phenolic compounds from pyrolysed biochars. For solid biochar, 10 g of biochar was poured into 150 ml of toluene and mixed for 7.5 h. The extracts were evaporated until the last drop of toluene was observed (usually takes 20–30 min) by using a rotatory evaporator (63 Heidolph, Germany). The residues left on the rotary evaporator were then poured into 10 ml acetonitrile. The obtained extracts were then filtered using a syringe filter (PTFE type, pore diameter 0.25 mm). The extracted solution was stored at 20 1C until further study. For tar sample, 10 ml of tar was diluted with acetonitrile, evaporated, filtered, and stored similarly as described above. Pyrolysis wood, biochar extract and tar were diluted 1:10 whereas pyrolysis hemp biochar was diluted 1:15 with acetonitrile before genotoxicity testing was conducted.
2.5. Characterstics of selected Ames strains The Salmonella typhimurium strains were maintained in frozen stocks ( 80 1C, DMSO culture) and grown as described by Maron and Ames (1983). Each strain was tested on the basis of associated genetic markers, raised from a single colony of the master plate (Maron and Ames, 1983). A set of five (TA97, TA98, TA100, TA102, and TA104) Ames strains was used; the characteristics of Salmonella typhimurium are given in Table S1 (Supplemental material). TA97, TA98, and TA100 cause mutation at G–C site whereas TA102 and TA104 causes mutation at A–T site. These sets of Ames strains were selected for this study due to their specific mode of action at G–C and A–T base pair at the critical sites of the reversion in the presence of different chemicals (carcinogens). TA98 and T100 strains are particularly sensitive to the mutagenicity of aromatic amines and polycyclic hydrocarbons. All strains are amino acid-dependent strains of Salmonella typhimurium each carrying different mutations in various genes in the histidine operon. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. In the absence of an external histidine source, cells cannot grow and form colonies. Only those bacteria that revert to histidine independence (his þ ) are able to form colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen (MMS as a positive control and tested biochar extract) was added to the plate, the number of revertant
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colonies per plate was increased, usually in a dose-related manner. Many mutagens and carcinogens found in the environment required metabolic activation to increase the sensitivity of the bacteria to the mutagenic effect of these compounds. Hence metabolic activation S9 was used in parallel with all test experiments. 2.6. Preparation of rat liver microsomal fraction Rat liver microsomal fraction (S9) was prepared according to the method of Garner et al. (1972).
lower than o2.1 and o 0.2 mg N g 1 biochar, respectively. In the field soil adsorption experiment, hemp biochar adsorbed 10.9 7 2.8 mg NH4þ –N g 1 biochar and 12.5 7 4.0 mg NO3 N g 1 biochar, whereas wood biochar adsorbed 4.8 72.1 mg NH4þ –N g 1 biochar and 3.8 71.6 mg NO3 –N g 1 biochar respectively (mean of three replicates). Total Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, W, and Zn toxic and non-esssential heavy metals concentrations were higher in hemp biochar as compare to wood biochar as shown in Table 1.
2.7. Salmonella mutagenicity testing The pre-incubation test was performed as described by Maron and Ames (1983) with minor modifications (Pagano and Zeiger, 1992). Five doses of hemp biochar extract (0.5, 1.0, 1.5, 2.0, and 2.5 ml per plate) and five doses of each wood biochar extract and tar (0.5, 1.0, 2.0, 4.0, and 8.0 ml per plate) were plated in triplicates with 0.1 ml of bacterial culture each. When the test sample and bacterial culture were incubated (30 min at 37 1C), 2.0 ml top agar containing traces of histidine and biotin were added. Contents were poured on minimal glucose agar plates. Plates were incubated at 37 1C for 48–72 h. Negative and positive controls were included in each assay. Negative control plates contained bacteria and acetonitrile solvent but no test sample. Methyl methane sulfonate and sodium azide (Maron and Ames, 1983; Anjum and Malik, 2013) served as positive controls. All extracts were also tested in the absence ( S9) or presence ( þS9) of rat liver microsomal fraction, to which 20 ml of S9 liver homogenate mix per plate was added. The criterion used to classify the results as positive was considered according to the studies of Vargas et al. (1995). 2.8. Statistical analysis Statistical analysis of the results of mutagenicity testing for the different parameters; mutagenic index (MI), mutagenic potential (M), induction factor (I) and analysis of variance (ANOVA) were carried out according to Anjum and Malik (2013). 2.8.1. Mutagenic index (MI) The number of his þ revertants in the sample was compared to the negative control by its mutagenic index value. Mutagenic index ¼
Number of his þ revertants induced in the samples Number of his þ revertants induced in the negative control
2.8.2. Mutagenic potential (M) The mutagenic potential of the test samples was calculated by the initial linear portion of the dose response curve with tester strains. The slope (m) was obtained by the least squares regression of the initial linear portion of the curve of initial dose response. 2.8.3. Induction factor (I) The induction factor for various test strains for different soil extracts was evaluated as follows: n c I ¼ ln c where n is the number of revertants in the samples and c is the number of revertants in the solvent control. The induction factor was calculated to determine the difference between two samples if the sensitivity pattern based on the slope (m). 2.8.4. Analysis of variance (ANOVA) To determine the significance of the number of his þ revertants in the samples compared to the control, one way ANOVA was done at Pr 0.05 using MINITAB Software, USA.
3. Results 3.1. Biochar characterization The total organic carbon of hemp biochar and wood biochar was 92.6% and 84.2% (mean of three replicates) and pH-KCl was 7.1 70.33 and 8.0 70.53 respectively. Nitrogen contents in biochar samples were lower than 4% resulting in high carbon and nitrogen ratio (4 478). The percentage of ash content in hemp biochar (13.1 70.1) was higher than wood biochar (4.2 70.2). NH4þ and NO3 concentrations in hemp biochar and wood biochar were
3.2. Determination of PAHs and phenolic compounds in pyrolysis hemp biochar, wood biochar and tar Gas chromatography and mass spectrometry analysis of biochars and tar samples revealed the presence of various PAHs. The total concentrations of PAHs in hemp biochar and wood biochar were 34.9 mg g 1 of dry mass and 33.7 mg g 1 of dry mass respectively as shown in Table 2; the concentration of PAH in the tar was considerably lower (17.4 mg g 1). Total phenolic compounds of 55 mg g 1 and 8.3 mg g 1 were also quantified in hemp and wood biochar, respectively. No phenolic compounds were detected in the tar sample. The analysis of hemp biochar revealed the presence of petrogenic PAHs i.e. napththaline, acetonaphthyene, acenaphthene and fluorine, whereas the concentrations of petrogenic PAHs (napththaline, acetonaphthyene, acenaphthene, and fluorine) in wood biochar were lower as compared to hemp biochar (Table 2). Pyrogenic PAHs, i.e. phenanthrene, anthracene, fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, and indeno (1,2,3-cd) pyrene were also determined in hemp biochar, however wood biochar revealed the presence of pyrogenic PAHs at a higher concentration as compared to hemp biochar (Table 2). It was observed that the concentration of pyrogenic and petrogenic PAHs in tar was lower than hemp biochar and wood biochar.
Table 1 Quantitative determination of toxic and non-esssential heavy metals in hemp biochar and wood biochar by ICP-OES analysis. Metals in sample
Al As B Ca Cd Co Cr Cu Fe K Mg Mn Mo Na Ni P Pb S Se W Zn
Concentration in dry biomass [mg g 1] Hemp biochar
Wood biochar
761 o 0.01 36.2 81,911 o 0.01 1.67 o 0.01 66.7 92.1 18,819 5705 565 o 0.01 2444 5.86 17,845 0.74 4768 1.56 o 0.01 528
72.4 o 0.01 1.58 1156 o 0.01 0.08 0.38 2.5 273 2944 428 23.7 0.15 326 0.28 763 0.35 362 0.07 o 0.01 12.2
Each value is the mean of three duplicates.
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Table 2 Quantitative determination of polycyclic aromatic hydrocarbons (PAHs) in pyrolysis biochars and tar by gas chromatography and mass spectrometry. Concentration in dry biomass [mg g 1]
PAHs (petrogenic and pyrogenic)
Naphthaline Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benz(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Dibenz(a,h)anthracene Benzo(g,h,i)perylene Total
Hemp biochar
Wood biochar
Tar (byproduct)
7.1 3.1 2.0 4.6 9.3 2.9 2.0 2.6 0.5 0.5 0.1 o0.05 0.1 o0.05 o0.05 o0.05 34.9
3.8 2.3 1.2 3.2 11 3.2 2.4 3.2 1.3 1.2 0.3 0.1 0.3 0.06 o 0.05 o 0.05 33.7
5.8 0.7 0.8 1.5 6.0 1.5 0.7 0.3 o0.05 o0.05 o0.05 o0.05 o0.05 o0.05 o0.05 o0.05 17.4
Each value is the mean of three duplicates.
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3.3. Reversion of the Salmonella tester strains in the presence of biochar and tar extracts Mutagenicity testing of biochars and tar was conducted by Ames tests. Five doses of hemp biochar extract (0.5, 1.0, 1.5, 2.0, and 2.5 ml per plate) and five doses of each wood biochar extract and tar (0.5, 1.0, 2.0, 4.0, and 8.0 ml per plate) were plated in triplicates with 0.1 ml of bacterial culture each. Positive and negative control of each experiment was run in parallel at the same five doses. Positive control (methyl methane sulfonate and sodium azide), biochars and tar extracts revealed reversion of Ames strains, whereas with negative control (acetonitrile solvent) at the same dose reversion in tester strains were not observed. Consequently, negative control test indicated that acetonitrile solvent was not mutagenic. The number of reverted tester strains are shown in Figs. 1–3 and reversion results (in terms of MI, I, m and LSD) of Salmonella typhimurium strains with hemp biochar, wood biochar and tar are summarized in Table 3. The data interpretation by statistical analysis in terms of mutagenic index, mutagenic potential, induction factor and analysis of variance indicates that biochar and tar samples exhibited maximum mutagenicity with TA98 and TA100 strains in the presence and absence of rat liver microsomal fraction (S9).
Table 3 Reversion of Salmonella tester strains in the presence of hemp biochar, wood biochar and tar in terms of mutagenic index (MI), induction factor (I), slope (m) of the initial linear dose response curve as determined by linear regression analysis and least significance difference (LSD). Salmonella strains
TA97 TA98 TA100 TA102 TA104 Salmonella strains
S9
Hemp biochar: mutagenic index (MI) 0.5 ml per plate
1.0 ml per plate
1.5 ml per plate
2.0 ml per plate
2.5 ml per plate
þ þ þ þ þ
2.7 2.7 8.5 8.3 2.0 2.3 1.1 1.6 1.5 1.5
4.0 3.9 10.0 12.0 2.7 3.3 1.3 2.0 1.6 1.8
4.5 4.7 14.8 15.5 3.1 4.0 2.1 2.2 1.8 2.0
5.0 5.4 15.3 17.0 3.5 4.3 2.3 2.7 2.1 2.2
3.7 4.3 12.2 13.3 2.7 3.6 1.9 2.1 1.7 1.7
S9
Wood biochar: mutagenic index (MI) 0.5 ml per plate
TA97 TA98 TA100 TA102 TA104 Salmonella strains
þ þ þ þ þ
1.1 1.2 1.8 1.9 1.2 1.4 1.1 1.2 1.0 1.0
S9
Tar: mutagenic index (MI) 0.5 ml per plate
TA97 TA98 TA100 TA102 TA104
1.0 ml per plate
þ þ þ þ þ
1.2 1.2 1.7 2.2 1.3 1.4 1.2 1.3 1.1 1.1
1.8 1.7 3.3 3.1 1.7 1.8 1.3 1.4 1.2 1.2
1.0 ml per plate 1.7 1.5 2.7 2.8 1.6 1.6 1.3 1.4 1.2 1.2
2.0 ml per plate 2.0 2.0 4.2 3.9 2.1 2.2 1.4 1.5 1.3 1.3
4.0 ml per plate 2.4 2.4 5.4 4.9 2.7 2.6 1.6 1.7 1.4 1.4
I
m
LSD P r 0.05
1.39 1.49 2.66 2.77 0.93 1.20 0.26 0.54 0.10 0.14
14.7 18.9 21.2 25.2 16.2 20.1 14.6 17.3 16.2 15.8
8.9 4.1 5.2 21.4 4.1 3.1 2.3 20.4 12.2 30.3
I
m
LSD P r 0.05
8.0 ml per plate 2.3 2.2 5.0 4.2 2.4 2.3 1.5 1.6 1.2 1.2
0.30 0.35 1.49 1.35 0.52 0.46 0.50 0.32 1.01 1.03 I
2.0 ml per plate 2.1 1.8 3.0 3.2 1.8 1.9 1.4 1.5 1.2 1.3
4.0 ml per plate 2.2 2.1 3.4 3.9 2.8 2.9 1.5 1.6 1.3 1.4
Abbreviations: S9, liver microsomal fraction; , in the absence of S9; þ , in the presence of S9.
3.5 3.7 3.9 3.8 5.7 5.6 3.3 4.4 2.9 2.8 m
9.7 8.8 4.8 4.8 2.5 5.5 2.9 12.4 3.6 8.3 LSD P r 0.05
8.0 ml per plate 1.9 1.8 2.8 3.1 2.0 2.0 1.1 1.2 1.0 1.1
0.25 0.13 0.89 1.09 0.36 0.37 0.60 0.40 1.16 0.97
2.7 2.9 1.8 2.7 4.9 4.3 2.6 3.3 2.5 3.1
17.5 12.2 4.5 12.7 5.0 3.2 2.8 14.4 4.3 5.7
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Fig. 1. Hemp biochar: number of reverted tester strains TA97, TA98, TA100, TA102, and TA104 in the absence ( S) or in the presence ( þS) of liver microsomal fraction on increased dose (0.5, 1.0, 1.5, 2.0 and 2.5 ml per plate) of hemp biochar. Control¼ without extract. * ¼ indicates maximum number of reverted tester strains.
Fig. 2. Wood biochar: number of reverted tester strains TA97, TA98, TA100, TA102, and TA104 in the absence ( S) or in the presence ( þS) of liver microsomal fraction on increased dose (0.5, 1.0, 2, 4.0 and 8.0 ml per plate) of wood biochar. Control¼ without extract. * ¼ indicates maximum number of reverted tester strains.
The number of reversion of tester strains increased in both presence and absence of S9 with an increasing dose of hemp biochar extract up to 2 ml per plate whereas a decline was observed at concentrations of 2.5 ml per plate (Fig. 1). Consequently, hemp biochar sample exhibit maximum mutagenicity with TA97, TA98 and T100 strains both in the presence and absence of S9 fraction. An increasing dose of test samples caused a gain of reverted tester strains (Fig. 1). While for wood biochar and tar extract a reversion of tester strains increased in the presence and absence of S9 with concentrations of extract up to 4 ml per plate whereas reversion was sharply decline at the dose of 8 ml per plate. Wood biochar and tar extract exhibited maximum mutagenicity with TA98 and TA100 strains in the presence and absence of rat liver (S9) microsomal fraction (Figs. 2 and 3). According to mutagenic index TA98 and TA100 was the most responsive strain against all test samples while other strains revealed weak response in comparison to the TA98 strain
Fig. 3. Tar: number of reverted tester strains TA97, TA98, TA100, TA102, and TA104 in the absence ( S) or in the presence ( þS) of liver microsomal fraction with increased dose (0.5, 1.0, 2, 4.0 and 8.0 ml per plate).
(Table 3). Highest mutagenic index value was observed for hemp biochar extract. Consequently, in terms of mutagenic index hemp biochar was the most mutagenic than the wood biochar and tar. The response of tester strains in terms of slope (m) of the initial linear dose response curve was obtained by the least squares regression. It was found that TA98 and TA100 showed the maximum slope value in hemp biochar extract. Only TA100 showed the maximum slope value in wood biochar and tar extract (Table 3). According to the observed value of induction factor (I), TA98 was the most responsive strain against all test samples while other strains revealed weak response in comparison to the TA98 strain (Table 3). However, biochar extracts and tar exhibited a variable trend in responsiveness of the different strains in the presence of S9 fraction in terms of slope with respect to the initial dose response curve. The significance of the reversion of tester strains with increasing doses was determined by one way ANOVA. The mutagenic potential of the hemp biochar, wood biochar, and tar extract was calculated by the initial linear portion of the dose response curve with tester strains. The mutagenicity for hemp biochar extracts in terms of mutagenic potential (M) of the initial linear dose response for the most responsive strain TA98 with and without S9 was higher as compared to wood biochar and tar. Net revertants per gram of hemp biochar, wood biochar and tar were 4,485,000, 522,000 and 387,500, respectively, for most responsive TA98 strains in the presence of S9 fraction. The maximum number of net revertants per gram of hemp biochar, wood biochar and tar in the absence of S9 fraction were 3,690,000, 435,000 and 310,000, respectively, for most responsive TA98 strains. The mutagenic activity was slightly enhanced in the presence of S9 for both biochar and tar extracts tested.
4. Discussion For the study of mutagenicity assessment of pyrolysis biochars, hemp biochar and wood biochar were produced by different types of feed stocks with same biochar production processes. The characteristics of hemp biochar and wood biochars were variably different from each other. We have been adopted Ames assay for the evaluation of risks associated with the handling and use of
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biochars. The result of this study reveals mutagenic potential of pyrolysis biochars. In our study we observed and discussed different mutagenic responses against biochars that are comparable to each other and also focused on the by-product tar. In the present study, characterized hemp biochar revealed higher adsorption of NH4þ –N g 1 biochar and NO3 –N g 1 biochar in the field soil as compared to wood biochar. On the other hand, ICP-OES analysis of hemp biochar and wood biochar for toxic and non-essential heavy metals revealed low concentrations of toxic heavy metals (As, Cr, Cd i.e. o0.01) as shown in Table 1, those may not have any toxic or mutagenic effect. In our study we have determined a combinations of petrogenic and pyrogenic PAHs i.e. napththaline, acetonaphthyene, acenaphthene, and fluorine; these were higher in hemp biochar as compared to wood biochar and tar. The concentration of pyrogenic PAHs i.e. phenanthrene, anthracene, fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, and indeno(1,2,3cd) pyrene were higher in wood biochar than hemp biochar. Here data of chemical analysis of biochars revealed the presence of greater abundance of pyrogenic PAHs as shown is Table 2. Comparable to wood biochar and tar, high mutagenicity was observed due to the presence of high concentration of petrogenic PAHs in hemp biochar. Hence, the results of this study indicate that petrogenic PAHs were more mutagenic than pyrogenic PAHs. Oleszczuk et al. (2014) collected soil samples in the vicinity of biochar production sites and detected polycyclic aromatic hydrocarbons (PAH). In their study, the concentrations of PAHs were 1796–101,282 μg kg 1, much higher than our study, however, temperature (400–500 1C) inside the kiln was same as in our study. Fabbri et al. (2013) also analyzed PAHs in biochar and biochar amended soil produced by slow pyrolysis from woody biomass, whereas we applied fast pyrolysis. Fabbri et al. (2013) reported a total PAH levels ranged between 1.2–19 μg g 1 and 0.2–5.0 μg g 1. Specifically, benzo(a)pyrene ranging between 0.01 and 0.67 μg g 1 across various biochars amended soils which were much higher than our biochar samples. Therefore, our data and other studies (Fabbri et al., 2013; Oleszczuk et al., 2014) indicate that some factors were influencing the production of mutagenic substances which was slow and fast pyrolysis process and temperature inside the kiln might be also a cause of production of mutagens (PAH). It can be assumed that pollutants of biochar might be overcome due to the further optimization of pyrolysis process. In addition, our results also suggest optimizing the pyrolysis to the type of biomass feedstock. The concentration of pyrogenic PAH was noticed much higher in hemp biochar as compared to wood biochar; and in mutagenicity testing, higher mutagenic potential of hemp biochar was observed as compared to wood biochar. Ames test by Maron and Ames (1983) is one of the important assays for testing of environmental samples. in vitro Salmonella/ microsomal assay with rat liver microsomal fraction (þS9) is also one of the most frequently used test for assessing the mutagenic potential for both pure compounds and complex mixture (Ames et al., 1975). An evidence of earlier study by Ames et al. (1975) revealed that fluorene and benzo(a)pyrene is a mutagen which has been detected in biochar samples (Table 2). In their study, 2-aminofluorene at the concentration of 2 mg per plate was mutagenic, and it acts as a direct mutagen which caused frame shift mutation at G–C site of TA98 and TA100 tester strains. Similar to Ames et al. (1975), in our study TA98 and TA100 tester strains were the most responsive tester strains. The concentrations of fluorine in hemp biochar and wood biochar were 4.6 mg g 1 and 3.2 mg g 1 respectively, whereas, concentrations of benzo(a)pyrene in hemp biochar and wood biochar were 0.18 mg g 1 and 0.37 mg g 1, respectively (Table 2). Maximum mutagenicity was observed in hemp biochar extract at
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the highest concentration of 2.0 ml per plate and observed 492 revertants in the absence of S9 and 598 revertants in the presence of S9 for most responsive TA98 strain can be observed in Fig. 1. Ames et al. (1975) also reported, when TA100 was treated with benzo(a)pyrene at concentration of 5 ml per plate and in the presence of S9 at concentration of 20 ml per plate, they observed 318 revertants per plate which is comparable to our study as 358 and 328 revertants per plate were obtained when TA100 was treated with wood biochar and tar (Figs. 2 and 3). Consequently our data indicates that pyrolysis biochars caused frame shift mutation at G–C site of TA98 and TA100 tester strains which revealed mutagenicity response due to detected PAHs in hemp biochar, wood biochar, and tar. Beside Ames Salmonella/microsomal mutagenicity test, genotoxicity assessment of biochar has employed many assays to detect DNA damage ability and mutagenicity such as Tradescantia micronucleus test and Microtoxs toxicity test ( Busch et al., 2013; Oleszczuk et al., 2014), and results of their studies are discussed here. Oleszczuk et al. (2014) performed several ecotoxicological tests (phytotoxicity to Lepidiumsativum, Microtoxs-Vibrio fischeri, MARA-11 different microorganisms, Daphtoxkit F™-Daphnia magna) to evaluate the effect of polluted soil by PAHs on plant, bacteria and crustaceans. Depending on the ecotoxicological assay applied and sampling sites, the toxicity of the soils was varied, but only in a few cases the phytotoxicity was correlated with the content of PAHs. Busch et al. (2013) tested genotoxic effect of different hydrochars and biochars with the Tradescantia micronucleus test and observed chromosomal aberrations in pollen cells of Tradescantia. These findings as above resulting from a completely different approach support our findings and underline the necessity of exhaustive risk assessment of biochars, namely of their processing and application.
5. Conclusion Only a few reports exist describing genotoxic impacts of biochar. As biochar production linked to mutagenicity and toxicity is a relatively new issue, this work provides first and new insights to this topic. In Ames Salmonella/microsomal mutagenicity test system, it was observed, that pyrolysis hemp biochar exhibited higher mutagenic effects than wood biochar and tar. As example, the Ames method clearly indicates that petrogenic PAHs and pyrogenic PAHs within the biochar caused frame shift mutation at G–C site of TA98 and TA100 tester strains. Consequently, our finding demands caution in handling of biochars, since the unknown mutagens may also impact on human health. Further research is urgently needed to optimize the biochar production process and to identify the unknown mutagens in biochars including their impact on biological systems.
Acknowledgments R.A. gratefully acknowledges the fellowship grant provided by the Leibniz Association and German Academic Exchange Service (DAAD, Grant no. A/12/93888). M.T.R. and N.K. are thankful for grants provided by the German Federal Ministry of Research and Education (BMBF, Grant no. 03SF0381A) and the German Federal Ministry of Food and Agriculture (BMEL, Grant no. 22026411). The authors thank T. Nohmi, National Institute of Hygienic Sciences, Division of Genetics and Mutagenesis, Tokyo, Japan, for the generous provision of Ames tester strains. Additionally, the authors would like to thank K. Mundt, T. Langer, U. Lüder and G. Franke for experimental support and C. Prautsch and L. Herkoltz for their support in analytical tasks. The author's sincere thanks
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