Biochar properties regarding to contaminants content and ecotoxicological assessment

Journal of Hazardous Materials 260 (2013) 375–382

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Biochar properties regarding to contaminants content and ecotoxicological assessment Patryk Oleszczuk ∗ , Izabela Jo´sko, Marcin Ku´smierz Department of Environmental Chemistry, Faculty of Chemistry, 3 Maria Curie-Skłodowska Square, 20-031 Lublin, Poland

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• Biochars from commercial manufacturer were analysed.

• Heavy metals and PAHs were analysed in context of ecotoxicological properties. • High concentration of PAHs was observed for some biochars. • Almost all tested biochars were toxic in bioassays applied.

a r t i c l e

i n f o

Article history: Received 25 February 2013 Received in revised form 1 May 2013 Accepted 24 May 2013 Available online 30 May 2013 Keywords: Biochar Polycyclic aromatic hydrocarbons Properties Ecotoxicity

a b s t r a c t The objective of the study was the determination of the content of contaminants and toxicity of four different biochars. The properties of the biochars, content of trace metals and polycyclic aromatic hydrocarbons (16 PAHs) were determined. Toxicological estimation of the biochars was performed on the basis of a battery of biotests with plants (Lepidium sativum), bacteria (Vibrio fischeri and 11 different strains from MARA), alga (Selenastrum capricornutum), protozoa (Tetrahymena thermophila) and crustaceans (Daphnia magna). The content of trace metals depended on the biochar and was comparable to uncontaminated soils. PAHs sum varied from 1124 to 28,339 ␮g/kg. The toxicity of the biochars depended both on their kind and on the test applied. The most sensitive organism was D. magna. Relatively the least sensitive to extracts from the biochars proved to be S. capricornutum and T. thermophila. A significant correlation between the content of PAHs and toxicity was noted only in the case of D. magna. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The application of biochar in soils is becoming an increasingly common treatment. Biochar is an interesting solution as on the one hand it improves the properties of soils [1,2], and on the other, which is extremely important, it may constitute a tool in the combat against the global worming through the sequestration of CO2 [3]. Recent studies showed, similarly to activated carbon (AC), that biochar may be use for the immobilisation of organic and inorganic contaminants in various environmental matrices, i.e. sediments,

∗ Corresponding author. Tel.: +48 81 5375515; fax: +48 81 5375565. E-mail address: [email protected] (P. Oleszczuk). 0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.05.044

soils and sewage sludges [4–6]. This results from the fact that biochar is characterised by high affinity to organic and inorganic contaminants [7]. Due to binding of contaminants by biochar, the mobility and bioavailablity of contaminants decrease, and thus also their potential toxicity to various groups of organisms. In spite of the positive effect that biochar may have on the soil, in the literature attention is paid more and more frequently on biochar contamination with polycyclic aromatic hydrocarbons (PAHs) and trace metals [8–11]. Depending on the kind of biochar, the content of PAHs may vary and exceed considerably the threshold values relating to biosolids [12]. It is assumed that both the concentration and the composition of individual PAHs depend to a notable extent on temperature and on the kind of biomass used to produce biochar [10,13]. The mechanism of the formation of PAHs in the

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process of pyrolysis has been studied in detail [10,14,15]. The PAHs formed in that process are then adsorbed on the biochar. Studies conducted by Ledesma et al. [14] and Masek et al. [15] suggested that PAHs are formed mainly at temperatures higher than 700 ◦ C. The latest research shows, however, that PAHs can also be formed at lower temperatures [10]. Next to the temperature another important parameter affecting the content of PAHs can be the kind of material used for biochar production [10,13]. The presence of PAHs in biochars puts, therefore, a question mark over the possibility of their uncontrolled utilisation in soil fertilisation. Another factor that may impose a considerable limitation on the utilisation of biochars is their content of trace metals. Studies concerned with the problem of trace metals are not as numerous as those relating to PAHs, but they also indicate that those compounds may be present in biochars [8,16], which creates a potential threat to the environment. The content of trace metals largely depends on their concentration in the initial materials used for the biochars production [16]. Both from a regulatory and environmental perspective, using a soil amendment material that contains PAHs and trace metals that pose a threat to the soil is unacceptable [11]. Studies conducted so far concerning biosolids show that chemical analysis are not a sufficient tool for the estimation of the risk related to the utilisation of these materials as a soil fertiliser [17]. Biological tests may prove to be useful for that purpose. Biotests should not be treated as an alternative to chemical analyses, but supplementation of studies with biological tests may expand the knowledge on the potential risks. In addition, the application of biological tests permits the study of the possible interactions among various contaminants that provide ultimate evidence on the existence or absence of toxic effect on organisms. The objective of the study presented herein was the estimation of the content of contaminants that may potentially occur in biochars, i.e. trace metals (Cd, Cu, Cr, Ni, Pb, Zn) and PAHs. The results obtained were related with ecotoxicological estimation covering a broad group of organisms (bacteria, plants, algae, invertebrates). The information acquired will permit the characterisation, as exhaustive as possible, of biochars with regard to their potential utilisation for soil amendment.

was produced of wheat straw and was provided by Mostostal Sp. z o.o. (Wrocław, Poland). The chemical properties of biochars were determined by standard methods. The pH was measured potentiometrically in 1 M KCl after 24 h in the liquid/soil ratio of 10. The cation exchange capacity (CEC) and available potassium, phosphorus and magnesium were determined according to procedures for soil analysis [18]. The amounts of carbon, hydrogen and nitrogen were determined using CHN equipment (Perkin–Elmer 2400). To analyse the textural characteristics of materials, low-temperature (77.4 K) nitrogen adsorption–desorption isotherms were recorded using a Micromeritics ASAP 2405 N adsorption analyser. The specific surface area SBET was calculated according to the standard BET method [19]. 2.2. Contaminants content analysis Pseudototal metal concentration was extracted in the PROLABO microwave oven (Microdigest 3.6, France), using a wet method in a mixture of nitric acid (8 ml) and perchloric acid (8 ml) at the ratio of 1:1. A 30% of hydrogen peroxide solution was added before acids. An analysis for the content of Cd, Cr, Cu, Fe, Ni, Pb, and Zn was carried out using emission spectrometry on ICP-OES (Leeman Labs, PS 950). Evaluation of the accuracy and precision of analytical procedures used reference materials (Heavy Clay Soil, RTH 953, Promochem). Polycyclic aromatic hydrocarbons were analysed according to the method developed by Hilber et al. [9]. Dry biochar samples were extracted using accelerated solvent extractor (ASE 200) from Dionex GmbH (Idstein, Germany). A qualitative and quantitative analysis of PAHs was carried out on a liquid chromatograph with DAD and Fluorescence detectors (Waters, USA). The procedural blank was determined by going through the same extraction and cleanup procedures for each series of samples. None of the analytical blanks were found to have detectable contamination of the monitoring PAHs and thus the results were not blank corrected. 2.3. Bioassays Biochars toxicity was assessed with the commercial toxicity bioassay – Phytotoxkit FTM Test [20]. The phytotoxkit microbiotest measures the decrease (or the absence) of seed germination and of the growth of the young roots after 3 days of exposure of seeds of selected higher plants to contaminated matrix in comparison to the controls in a reference soil. The analyses and the length measurements were performed using the Image Tool 3.0 for Windows (UTHSCSA, San Antonio, USA). The bioassays were performed in three replicates. Biochar was added to OECD soil at three doses 1%, 5% and 10%. To evaluate the effect of biochars on alga, bacteria, protozoa and crustaceans extracts from biochars were tested. Extracts were obtained according to the EN 12457-2 protocol [21]. The samples were mixed with de-ionised water in a single-stage batch test performed at a liquid-to-solid (L/S) ratio of 1 l/100 g. The glass bottles

2. Materials and methods 2.1. Biochar characteristic Four different biochars (Table 1) were used in the present study. All the biochars investigated here were obtained from commercial manufacturer and were produced by pyrolysis where the feedstock is thermochemically decomposed at a temperature range from 350 ◦ C (start of combustion) to 650 ◦ C (max. combustion temperature) in an oxygen-poor atmosphere (1–2% O2 ). Biochars BC-M, BC-O and BC-W were produced of elephant grass (miscanthus), coconut shell and wicker, respectively and were provided by com˛ Poland). Biochar BC-2 mercial manufacturer Fluid S.A. (Sedziszów, Table 1 The physico-chemical properties and elemental composition of investigated biochars. Biochars

BC-2 BC-W BC-O BC-M

pH

9.9a 9.1a 8.0b 6.8c

CEC

530a 143b 148b 144b

Available forms of P2 O5

K2 O

540a 122b 106a 132b

2824a 772b 1456c 1556c

Elemental composition Mg 163a 32b 156a 39b

C

H

N

53.87a 69.58b 62.89b 69.50b

1.76a 3.24b 2.41c 3.11b

0.91a 0.79b 0.93a 0.62c

H/C

SBET

0.033a 0.046b 0.038a 0.045b

26.3a 11.4b 3.1c 0.76d

pH in KCl. CEC – the cation exchange capacity (mmol/kg); P2 O5 , K2 O and Mg – available forms of phosphorous, potassium and magnesium (mg/kg); CHN – contribution (%) of carbon, hydrogen and nitrogen; H/C – ratio of hydrogen to carbon; SBET – specific surface area (m2 /g). Different letters means statistically significant differences (P ≥ 0.05) between biochars.

P. Oleszczuk et al. / Journal of Hazardous Materials 260 (2013) 375–382

were shaken in a roller-rotating device at 10 rpm. The extracts were filtered by filter with a porosity of 0.45 ␮m. A battery of 5 bioassays was used for study: Microtox, Microbial Assay for toxic Risk Assessment (MARA), Algaltoxkit FTM , Protoxkit FTM and Daphtoxkit FTM . Microtox reagents were purchased from SDI (Delaware, USA), MARA reagents from NCIMB Ltd. (Aberdeen, UK) while microbiotests (Algaltoxkit FTM , Protoxkit FTM and Daphtoxkit FTM ) with all reagents and equipment were purchased from MicroBioTests (Creasel, Deinze, Belgium). The Microtox® Toxicity Test was used to evaluate the inhibition of the luminescence in the marine bacteria Vibrio fischeri according to the test protocol [22]. The tests were carried out using a Microtox M500 analyser. The light output of the luminescent bacteria from biochars extract was compared with the light output of a blank control sample. Luminescence inhibition of extract was assessed for 15 min of exposure carrying out the “81.9% Basic test protocol” (screening test, MicrotoxOmni software was used). Microbial Assay for Risk Assessment (MARA) is a multi-species assay which allows measurement of toxic effects of chemicals and environmental samples. The test uses a selection of taxonomically diverse microbial species lyophilised in a microplate. Ten prokaryotic species and a eukaryote (yeast) constitute the biological indicators of toxicity assessment. In the present work, the microbial species used consisted of ten bacterial species: 1. Microbacterium sp., 2. Brevundimonas diminuta, 3. Citrobacter freundii, 4. Comamonas testosteroni, 5. Enterococcus casseliflavus, 6. Delftia acidovorans, 7. Kurthia gibsonii, 8. Staphylococcus warnerii, 9. Pseudomonas aurantiaca, 10. Serratia rubidaea, and one yeast species, 11. Pichia anomalia [23]. The growth of the organisms exposed to the test sample is determined with the reduction of tetrazolium red (TZR). A scanned image of the microplate obtained using a flatbed scanner is analysed using purpose-built software. The MARA test was performed according to the standard protocol described by Wadhia et al. [23]. Algaltoxkit FTM uses the green microalgae Selenastrum capricornutum, de-immobilised from alginate beads, for determination of algal growth inhibition after 72 h. Tests were performed according to the standard operational procedure manual of the Algaltoxkit FTM [24], which follows OECD Guideline 201 [25]. The algal biomass was evaluated by measuring the optical density of algal suspensions at 670 nm after 72 h of exposure to extracts or control solutions. Protoxkit FTM test measures growth inhibition of the ciliate protozoan Tetrahymena thermophila after 24 h. The tests were performed according to the standard operational procedure manual of the Protoxkit FTM [26]. Daphtoxkit FTM acute test with Daphnia magna makes use of neonates hatched from dormant eggs (ephippia) to determine the inhibition of motility after 24 h. Tests were performed according to the standard operational procedure manual of the Daphtoxkit FTM magna [27], which follows OECD Guideline 202 [28]. In order to check the correct performance of the tests and the sensitivity of the test organisms, a quality control test was performed with the reference chemical potassium dichromate (K2 Cr2 O7 ). The whole procedure was carried out according to user’s manual. The quality control test was successful.

2.4. Data analysis Toxicity data were expressed as the percentage of toxic effect (PE) compared to the control. The differences between each treatment and the control as well as between treatments were evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test.

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Table 2 Heavy metals content (mg/kg) in investigated biochars. Heavy metals

BC-M

BC-O

BC-W

BC-2

Cd Cu Ni Pb Zn Cr

0.87a 2.22a 9.95 22.3a 102.0a 18.0a

0.10b 3.81b nd 23.7a 30.2b 1.3b

0.20b nd nd 20.6a 97.9a nd

0.04c nd nd 21.6a 32.9b nd

nd – not detected. Different letters means statistically significant differences (P ≥ 0.05) between biochars.

3. Results 3.1. Biochar properties Table 1 presents the properties of the biochars studied. Almost all biochars showed an alkaline character (pH 8.0–9.9). The exception was the biochar derived from miscanthus (BC-M), for of which pH was at the level of 6.8. The highest values of CEC were noted for BC-2 (530 mmol/kg) which was obtained from another manufacturer than three other biochars. For these biochars the values of CEC were several-fold lower than for BC-2 and ranged from 143 to 148 mmol/kg. The highest levels of available forms of P2 O5 , K2 O and Mg were also noted, as in the case of CEC for biochar BC-2. However, significant differences between P2 O5 , K2 O and Mg were observed among the particular biochars from the same manufacturer (BC-W, BC-M and BC-O). Irrespective of the kind of biochar, the highest content was noted for K2 O (772–28241 mg/kg). Only BC-O was characterised by a higher level of Mg than of P2 O5 . In the remaining cases, the content of P2 O5 was significantly higher than that of Mg. The elemental analysis (CHN) indicated relatively small differences among the biochars. The highest level of carbon in the samples tested was characteristic of BC-W and BC-M. In the case of those biochars also the highest level of H was observed. The content of nitrogen was the highest in BC-O and BC-2. Molar ratios of elements (H/C ratio) were determined to estimate the aromaticity and carbonisation of biochars. All tested biochars were characterised by a very low H/C ratio confirming a high level of carbonisation and aromatisation of these materials [29] (Table 1). The degree of carbonisation of the biochars studied was as follows: BCW < BC-M < BC-O < BC-2 and did not differ significantly among the particular biochars. Whereas, the biochars differed notably from one another in terms of their surface area (SBET ). The biochar with the highest specific surface area was BC-2. Values smaller by more than a half were noted for BC-W, while BC-O and BC-M had the lowest values of SBET . 3.2. Trace metals content The content of trace metals was distinctly related with the kind of biochar. Only in the case of Pb the occurrence of that element was found to be at a similar level with no significant differences among the particular biochars (from 20.6 to 23.7 mg/kg). In the case of the remaining metals notable differences were observed. In ascending concentration these were: Cd (0.04–0.87 mg/kg), Cu (0.00–3.81 mg/kg), Ni (0.00–9.95 mg/kg), Cr (0.00–18 mg/kg) and Zn (30.2–102.0 mg/kg) (Table 2). The biochar derived from miscanthus (BC-M) was characterised by the highest levels of Cd, Ni, Zn and Cr. The content of Cd in BC-M was several-fold higher comparing to the remaining biochars. The biochar derived from coconut (BC-O) was characterised by the highest content of Cu. The lowest frequency of occurrence in the biochars studied was noted for Ni. The presence of Ni was observed only in biochar BC-M. Estimating the content of trace metals in relation to the Polish and EU norms concerning sewage sludge (Table S1), in no case their

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Concentration [ng/g]

700

700

BC-W

BC-O

PAHs sum = 1124.2 ng/g

PAHs sum = 1223.5 ng/g

600

600

500

500

400

400 300

300

200

200

100

100 0

0 Na

Ace

Ac

Fl

Phen Ant Fluo Pyr BaA

Ch

BbF BkF BaP

Na

Ace

Ac

Fl

PAHs

Ch

BbF BkF BaP

PAHs

14000

3000

BC-M

BC-2

PAHs sum = 28339.1 ng/g

12000

Concentration [ng/g]

Phen Ant Fluo Pyr BaA

PAHs sum = 7155.7 ng/g

2500

10000

2000

8000 1500 6000 1000

4000

500

2000 0

0 Na

Ace

Ac

Fl

Phen Ant Fluo Pyr BaA

Ch

BbF BkF BaP

PAHs

Na

Ace

Ac

Fl

Phen Ant Fluo Pyr BaA

Ch

BbF BkF BaP

PAHs

Fig. 1. Polycyclic aromatic hydrocarbons content in biochars used in the experiment. Error bars represents standard deviation error (SD, n = 3 extractions).

exceeding was noted. The assayed levels of the metals were considerably lower than the permissible norms, which may suggest an absence of threat on their part under conditions of biochars utilisation as a soil fertiliser. 3.3. Polycyclic aromatic hydrocarbons content Fig. 1 presents the content of PAHs in the biochars studied. The level of the sum of PAHs clearly depended on the kind of material tested. The highest concentration of the sum of PAHs was noted for biochar from miscanthus (BC-M) (28 339 ␮g/kg). A relatively high level of PAHs was also found in the case of the straw-derived biochar (BC-2). In this case the content of PAHs was at the level of 7156 ␮g/kg. The content of the sum of PAHs in the two remaining biochars, i.e. BC-W and BC-O, did not differ significantly from each other and amounted to 1124 ␮g/kg and 1224 ␮g/kg, respectively. The dominant group of PAHs in terms of their content were 3-ring compounds which constituted depending on the biochars from 64.6% to 82.6% of total PAHs content. Within 3-ring PAHs the notably dominant compound was phenanthrene (28.7–53.1%). Other compounds with a high contribution were fluorene (11.3–26.8%), anthracene (5.2–13.4%) and pyrene (5.0–13.8%). In none of the biochars studied was presence detected of the heaviest PAHs, i.e. DahA, BghiP and Ind.

between the dose and the effect observed. A stimulating effect was also noted for the biochar derived from coconut (BC-O). As it was observed for BC-W, also in this case no significant relation was found between the dose and the effect. The highest dose of BCO, however, caused a slight inhibition of root growth, at the level of about 4%. Relatively the most unfavourable effect of the biochars under study was noted for the biochar derived from miscanthus (BC-M) and from wheat straw (BC-2). Only the lowest doses (1%) of those materials did not cause any significant inhibition of root growth. Increase of the doses of both BC-M and BC-2 to 5% caused a significant increase in their toxicity that did not differ significantly between the materials and amounted to 48% and 33%, respectively. The highest doses of BC-M and BC-2 (10%) caused further increase

3.4. Ecotoxicological properties The ecotoxicological estimation of the biochars studied depended clearly both on the test applied and on kind of biochar tested (Figs. 2–5). In none of the cases was any significant effect of the materials studied on seed germination found (data not shown). Whereas, significant differences between the particular biochars were observed for root growth inhibition (Fig. 2). The lowest toxicity towards Lepidium sativum was characteristic of the biochar derived from wicker (BC-W). Irrespective of the dose applied it stimulated root growth at the level of 12–41% with relation to the control OECD soil. However, no significant relationship was noted

Fig. 2. Effect of biochars on root growth inhibition of L. sativum depending on their dose. Values are given as averages with standard deviations (n = 3).

P. Oleszczuk et al. / Journal of Hazardous Materials 260 (2013) 375–382

c

Luminescence inhibition [%]

100

d 80

60

a 40

20

b

0 BC-W

BC-O

BC-M

BC-2

Biochars Fig. 3. Inhibition of V. fischeri luminescence after exposure to biochars’ leachates. Values are given as averages with standard deviations (n = 3).

of toxicity which for BC-M attained nearly 92%. The toxicity of BC-2 was not as high as that of BC-M and was at the level of 58% (Fig. 2). As in the case of the Phytotoxkit FTM test, also in the case of bacteria significant differences between the biochars under study were observed (Figs. 3 and 4). Greater differentiation among the particular biochars being noted in the case of the test Microtox® (Fig. 3) than of the MARA test (Fig. 4). In the Microtox® test all biochars studied showed significant inhibition of luminescence of

V. fischeri. The strongest negative effect, as in relation to L. sativum, was observed for BC-M. In the case of that biochar, luminescence inhibition was at the level of 99%. Only a slightly lower value (85%) was characteristic of biochar BC-2. For the remaining two biochars, i.e. BC-W and BC-O, inhibition of luminescence of V. fischeri was not as strong (Fig. 3) and was at the level of 40% and 12%, respectively. In the MARA test, an evidently negative impact with relation to all the species was observed only for BC-O (Fig. 4). Biochars BC-M and BC-2 also showed a toxic character towards more than a half of the test organisms. In the remaining cases, the values did not differ significantly in relation to the control, or had a slight stimulating effect on the growth of the test microorganisms (BC-M – C. stosteroni, BC-2 – Microbacterium sp.). Biochar BC-W was the only one to display a distinctly positive impact and no differentiation among the test species (Fig. 4). The sensitivity of the particular test organisms to the extracts depended also on the kind of material and was closely related with the kind of biochar applied. The MARA assay generates a Toxic Unit (TU) for each of 11 microbial species exposed to biochar’s extract. The TU for the particular biochars were as follows: BC-W < BC-2 < BC-M < BC-O. In accordance with the principle of the MARA assay, biochars with similar fingerprints may display similarities in their toxic effects. The dendrogram resulting from cluster analysis of values determined for each biochar is able to discriminate their (dis)similarities based on toxic fingerprinting (Fig. S1, electronic annex). BC-M, BC-O and BC-2 where closely clustered, which may suggest similar toxic effects, as opposed to BC-W which indicates that it toxicity manifests differently to other tested biochars. Fig. 5A and B presents the toxicity of extracts from biochars with relation to S. capricornutum (Algaltoxkit FTM ) and Tetrahymena thermophila (Prototoxkit FTM ). In both cases the lowest toxicity was characteristic of biochar BC-M, no negative effect of that biochar on the test organisms being found. The effect of the remaining

160

160 BCO Growth relative to control row [%]

Growth relative to control row [%]

BCW 140 120 100 80 60 40 20 0

140 120 100 80 60 40 20 0

1

2

3

4

5

6

7

8

9

10 11

1

2

3

4

5

Strains

6

7

8

9

10 11

7

8

9

10 11

Strains

160

160 BCM

BC2 Growth relative to control row [%]

Growth relative to control row [%]

379

140 120 100 80 60 40 20 0

140 120 100 80 60 40 20 0

1

2

3

4

5

6 Strains

7

8

9

10 11

1

2

3

4

5

6 Strains

Fig. 4. Effect of biochars’ leachates on microorganisms in MARA bioassay. Values are given as averages with standard deviations (n = 3).

P. Oleszczuk et al. / Journal of Hazardous Materials 260 (2013) 375–382

25

A

a

30

100

B

20 25 20 15

b 15 10 a

10

b

c

0 BC-O

BC-M

40

d a

c

0 BC-W

c

60

20

5 5

C

80

b

a

Mortality [%]

Growth inhibiton [%]

35

Mortality [%]

380

BC-2

BC-W

BC-O

Biochars

BC-M

Biochars

b

0 BC-2

BC-W

BC-O

BC-M

BC-2

Biochars

Fig. 5. Toxicity of raw (A – AlgaltoxkitFTM , B – ProtoxkitFTM ) and diluted leachates (C – DaphtoxkitFTM ) from tested biochars.

materials clearly depended on the kind of assay. The highest toxicity towards S. capricornutum was characteristic of BC-O and BC-W (no significant differences between those biochars) which caused mortality at the rates of 26.5% and 21.0%, respectively. The toxicity of BC-2 with relation to S. capricornutum was relatively low (at only 2%). As in the case of S. capricornutum, also with relation to T. thermophila the strongest toxicity was characteristic of biochar BC-O (mortality at the level of 16.8%). A similar rate of mortality, 15.1%, was noted with relation to BC-2. The toxicity of BC-W with relation to T. thermophila was lower by more than a half compared to BC-O and BC-2. The most sensitive organism to the extracts from biochars was crustaceans – D. magna. Raw extract obtained from BC-W, BC-M and BC-2 caused 100% mortality of those test organisms (Fig. S2, electronic annex). Only in the case of BC-O relatively low values were observed, at the level of 10%. Double dilution of the extract did not produce any effect in the case of BC-M and BC-2, as the rate of mortality observed was still 100% (Fig. S2). Only 10-fold dilution of the extract permitted a differentiation in the results obtained (Figs. 5C and S2). The results obtained revealed the same trend that was observed in the case of V. fischerii. The toxicity of the particular biochars was as follows BC-O < BC-W < BC-2 < BC-M. 4. Discussion Up to date no research has been conducted on the toxicity of biochars. The available literature data relate primarily to the content of polycyclic aromatic hydrocarbons [8,9,11,13] and, to a lesser extent, trace metals [8]. The results obtained indicate that the content of trace metals may differ significantly among the biochars studied. However, both their level and thus the potential threat of accumulation in soils during the application of biochars should not constitute any real danger to living organisms (Table 2). Taking into account the level of biochars contamination with trace metals, their addition to soil should not cause any significant increase in their content in the soil thus amended. The assayed values of the content of trace metals were at a level similar to data presented by other authors [8]. Relatively higher levels of trace metals were noted for one of the biochars–that derived from miscanthus (BC-M) (Table 2). Miscanthus is frequently used as an energy crop and grown in soils fertilised with sewage sludge or wastewaters. Research shows that the plant displays a high capability of accumulating trace metals from soils [30], which could have been related with its higher content of those contaminants compared to the remaining materials tested. It should be emphasised, however, that its content of metals, though higher than in the other biochars under study, was still significantly lower than the permissible norms [31], and also lower than their average content in sewage sludges and composts [8]. The

situation was totally different in the case of PAHs. Their content varied within a very broad range (Fig. 1), considerably exceeding the acceptable level [12] in the case of two biochars: BC-M and BC-2. The problem of biochar contamination by PAHs was deeply investigated by Hale et al. [11]. Over 50 biochars were analysed and the total PAHs concentrations ranged from 70 to 3270 ␮g/kg. A similar level of the sum of PAHs (1480–5480 ␮g/kg) was also observed by Cang et al. [32]. In a study conducted by Freddo et al. [8] it was found that the content of the sum of PAHs may vary, depending on the kind of biochar, within the range from 80 to 8700 ␮g/kg. Distinctly higher levels of the sum of PAHs in biochars were noted by Hilber et al. [9] and Keiluweit et al. [10]. The values assayed by those authors varied from 9110 to 355,300 ␮g/kg (sum of 16 PAHs), and from 30,500 to 50,000 ␮g/kg (sum of total PAHs and their methylated derivatives). In the latter study the authors demonstrated a distinct relation between the content of PAHs and temperature of pyrolysis. The highest levels of PAHs were noted in biochars produced at temperature of 500 ◦ C, i.e. lower than that applied in this study. It should be emphasised that in a majority of the cases cited [8,10,11] biochars were obtained under controlled laboratory conditions. Only in the study by Hilber et al. [9] all biochars studied by those authors originated from commercial suppliers, like the biochars used in this study. This may suggest that biochars obtained under industrial conditions are characterised by PAHs levels higher those in biochars obtained in a laboratory. This finds support in the study by Freddo et al. [8], where PAHs level in biochar obtained from a commercial manufacturer was distinctly higher than in biochars produced by the authors under controlled laboratory conditions. The composition of the particular PAHs in the biochars under study was also more similar to the results presented by Hilber et al. [9] and by Keiluweit et al. [10] than to the remaining studies cited. Predominant in the biochars were 3-ring PAHs, primarily phenanthrene. Whereas, on most of the biochars no presence of naphthalene was detected. Like in the study by Freddo et al. [8], no content of 6-ring PAHs was found. However, the PAHs composition in biochars depended to a notable extent on the kind of material from which the biochars were produced, which may explain the observed differences with relation to the studies by other authors. This results support the hypothesis that the conditions under which pyrolysis is conducted are also important. At present there are no established norms concerning the permissible levels of PAHs in biochars applied to soils. Solutions proposed by the EU assume that the level of the sum of total PAHs in biochars should not exceed, depending on the proposal, from 6000 to 20,000 ␮g/kg [9]. At present biochars can be compared to biosolids applied to soils, for which established norms already exist. Maximum allowable concentrations for biosolids considered for the application to agricultural land are 6 mg/kg according to EU

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100

D. magna mortality [%]

80

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40

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0 0

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may pose a real threat. Especially in the case of one biochar the assayed levels of PAHs notably exceeded the permissible limits established for biosolids. Nevertheless, among the PAHs analysed no presence of 6-ring compounds was observed which are relatively stable and characterised by mutagenic and carcinogenic properties. The level of PAHs in the biochars was relatively high, which may – after the application of the biochars to soils – significantly increase their content. This may constitute a threat to organisms, which was confirmed by the ecotoxicological tests. The literature does not provide information on the subject of recommendations concerning the dosage of biochars. Considering the results obtained it should be emphasised that biochars, like other fertilisers, should be subject to strict control and should not be applied to soils without prior analyses. Of particular concern is the negative effect of biochars on the ecotoxicological parameters tested. Although there are no official norms concerning the application of biotests in the evaluation of biosolids, the results obtained indicate a risk of their negative effect on biological life. Biochars are usually applied in larger amounts than is the case with biosolids. In this situation, norms concerning the content of PAHs and trace metals in biochars should take that problem into account.

Fig. 6. The relationship between sum of 16 PAHs and mortality of D. magna.

Acknowledgments proposal (for the sum of 11 PAHs) [12]. Only in the case of one biochar its level of PAHs would disqualify it from agricultural use (Fig. 1). This is a very favourable result for the current situation of growing interest in the use of biochars. However, it indicates a need for research in this area, for the purpose of avoidance of potential threats. Chemical analyses are common in the estimation of the applicability of various materials. However, only the use of biological tests makes it possible to better identify the potential risk involved in the application of a given material for fertilisation purposes [17]. The effect of the biochars studied on toxicity towards the test organisms was varied but, as can be assumed on the basis of the results obtained, closely related with the content of certain polycyclic aromatic hydrocarbons (Fig. 6 and Table S2b – electronic annex). The most sensitive organisms to the biochars proved to be D. magna (Fig. 6). Whereas, the biochars inhibited the growth of algae and protozoa at levels not exceeding 20% and 25%, respective (in the worst case). The literature does not provide any data on the toxicity of biochars with relation to various organisms. Studies concerning other materials that can potentially play the role of fertilisers are also limited. Relatively the most frequently applied are tests concerning phytotoxicity [33] and tests with leachates from biosolids [34]. The levels of toxicity obtained for the biochars under study were similar to those observed for biosolids [17,35]. This indicates that biochars may have a significant effect on biological life also in the negative sense. For the purpose of estimation of the effect of the particular contaminants on the toxicity of the biochars studied analysis of correlation was performed (Table S2). A significant correlation between the toxic effect and the content of contaminants was noted only in the case of the sum of total PAHs (and selected compounds) and the rate of mortality of D. magna (Fig. 6). 5. Conclusion To our knowledge, the results presented here are the first study where the ecotoxicological properties of different biochars were determined in the context of organic and inorganic pollutants. While in the case of trace metals one should not expect any significant negative effect on the environment after the introduction of biochars to soils, the content of polycyclic aromatic hydrocarbons

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