Aged biochar changed copper availability and distribution among soil fractions and influenced corn seed germination in a copper-contaminated soil

Chemosphere 240 (2020) 124828

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Aged biochar changed copper availability and distribution among soil fractions and influenced corn seed germination in a coppercontaminated soil
ria Silva Gonzaga a, *, Maria Iraildes de Almeida Silva Matias b, Maria Isido Kairon Rocha Andrade a, Amanda Nascimento de Jesus a, Grazielle da Costa Cunha c, **, Raquel Santos de Andrade a, Jose Carlos de Jesus Santos a ~o Cristo ~o, SE, 49100-000, Brazil
va Agronomy Department, Federal University of Sergipe, Sa Soil Science Department, Federal Institute Baiano, Valença, BA, 45400-000, Brazil c ~o Cristo ~o, SE, 49100-000, Brazil
va Chemistry Department, Federal University of Sergipe, Sa a

b

h i g h l i g h t s
Biochar reduced the most available fractions of Cu in the contaminated soil.
Copper associated with organic matter was increased in biochar soil.
Seed germination was improved with coconut and orange biochar.
Aged-sewage sludge biochar still reduced seed germination.
Vigorous root and plantlet development occurred with sewage sludge biochar.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 April 2019 Received in revised form 8 July 2019 Accepted 9 September 2019 Available online 12 September 2019

Biochar has been recommended as a multi-beneficial amendment for the in situ remediation of heavy metals contaminated soils due to its high recalcitrance, stability, specific surface area and retention capacity, which leads to a long-lasting influence on the immobilization of soil contaminants. The influence of biochar on the availability of heavy metals such as copper is not fully understood and may be related to a change in copper association with soils fractions. Therefore, a long-time laboratory incubation study was set up as a completely randomized design to test the effect of biochar from different sources (coconut husks-CHB, orange bagasse-OBB and sewage sludge-SSB) at two rates of application (30 and 60 t ha1) on the distribution of copper in a copper-contaminated soil after 24 months incubation. Copper distribution was evaluated through a sequential extraction procedure that fractionated copper into five fractions: F1 (soluble and exchangeable), F2 (specifically bound), F3 (organic matter bound), F4 (Fe and Mn oxide bound) and F5 (residual). Copper availability, soil pH and organic matter were also evaluated. Corn seeds were germinated in the incubated biochar soil to investigate the effect of biochar on seed germination and plantlets characteristics. All biochars increased soil pH and the concentration of oxidizable organic matter, and reduced copper availability after the 24 months incubation. CHB caused a discrete influence on copper distribution among soil fractions. OBB30 increased F1 (54.5%), F3 (24.0%), F4 (32.2%) and F5 (64.1%), and reduced F2 (39.8%); OBB60 reduced F1 (61.8%), F2 (16.5%) and F3 (16.0%) and increased F4 (18.0%) and F5 (84.4%). SSB30 strongly reduced Cu concentration in F1 (96.2%), F2 (34.0%), and F3 (22.2%), and increased F4 (54.4%); SSB60 reduced F1 (57.5%) and F3 (59.4%). Considering the high stability of biochar, the association of copper to the organic fraction leads to a long-time reduction in copper availability in the contaminated soil, which can reduce the cost and increase the efficiency of the remediation process. SSB reduced seed germination but produced vigorous and well-developed plantlets. Therefore, with proper production procedure to reduce the volatile matter content, SSB may not interfere

Handling Editor: Patryk Oleszczuk Keywords: Crop residues Sewage sludge Biochar Copper Bioavailability

* Corresponding author., ** Corresponding author. E-mail addresses: [email protected] (M.I.S. Gonzaga), grazy.ufs@gmail. com (G.C. Cunha). https://doi.org/10.1016/j.chemosphere.2019.124828 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

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M.I.S. Gonzaga et al. / Chemosphere 240 (2020) 124828

with seed germination and has the greatest potential to be used for the remediation of coppercontaminated sites. © 2019 Elsevier Ltd. All rights reserved.

1. Introduction Copper is an essential plant micronutrient required in small concentrations in many important metabolic reactions, including photosynthesis and protein synthesis. However, elevated levels of copper in soil pose toxicity risk to humans, animals and plants (Kabata-Pendias, 2011). Seed germination, for instance, is strongly affected by high concentration of copper in soil. Wheat seed germination was reduced in 40% (Singh et al., 2007) and 46% (Gang et al., 2013) in a 100 ppm Cu-contaminated soil. Germination of rice seeds was reduced in 60% when exposed to 10 mM Cu (Mahmood et al., 2007). Other studies also reported deleterious effect of excess copper in soybean and chickpea seed germination (Adhikari et al., 2012). Therefore, crop productivity can be seriously impaired by copper toxicity. Copper contamination in agricultural soils is very common and results from the frequent use of fertilizers and pesticides to increase crop production and to reduce the incidence of pests and diseases. Copper-based fungicides are the major source of Cu contamination in many organic-based farms and vineyards (Meier et al., 2017). Remediation of copper contaminated agricultural soils is of paramount importance in order to avoid contamination of the food chain and maintain crop productivity. The immobilization of copper in soil is a cost-effective in-situ remediation technique that has been successfully used in many parts of the world. It is based on the capacity of the copper ion to bind with soil components as well as with soil amendments with high binding capacity. Copper solubility in soil is greatly dependent on soil texture, pH and organic matter (Bravin et al., 2012; Adrees et al., 2015). Soil pH bellow 6 increases copper availability (Adriano, 2001) due to an increase in positive variable charges in soil, while soil organic matter decreases copper availability due its high affinity for copper on the surface of functional groups (Bakshi et al., 2014). Copper adsorption to organic matter increases with soil pH due to the increase in the number of soil negative charges. Organic binding sites could be in the humic as well as in the dissolved fraction, which strongly influence copper adsorption efficiency due to the variable degree of decomposition of different organic fractions (Wang et al., 2015). In addition to organic matter, other soil components can also associate with copper and change its chemical-physical forms including exchangeable fraction, FeeMn oxide bound fraction, carbonate-bound fraction and residual fraction. Biochar, a recalcitrant organic material from the carbonization of organic residues, has been recently reported as an efficient tool for the remediation of contaminated soils due to its stability and ability to improve soil properties and reduce the availability of heavy metals (Cao and Harris, 2010; Mendez et al., 2012; Fang et al., 2016; Tomczyk et al., 2019). The many surface functional groups and adsorption sites on biochar can reduce copper availability in contaminated soils (Paz-ferreiro et al., 2014; Bakshi et al., 2014;

Zhou et al., 2017) and the deleterious effect of excess copper in plants. Moreover, most biochar has the potential to increase soil pH due to its alkaline nature (Li et al., 2016; Zhou et al., 2017), which also can increase copper adsorption. However, information on the effect of biochar from coconut shell, orange bagasse and sewage sludge on the distribution of copper among soil fractions is still scarce. Even though many studies have reported beneficial effects of biochar on seed germination (Free et al., 2010; Solaiman et al., 2012; Liopa-Tsakalidi and Barouchas, 2017), some biochar such as those of sewage sludge (Gonzaga et al., 2017a, 2017b), wood, paper
et al., 2016) and corn cobb (Intani et al., 2019) can also mill (Gasco cause adverse effect on seed germination probably due to high volatile matter content and soluble phytotoxic substances. Addition of biochar to copper-contaminated soils could further impact seed germination and needs more investigation. Therefore, this study aimed to evaluate the effect of different types and rates of application of aged biochar on the availability and distribution of copper in a contaminated soil using a sequential extraction procedure. The effect of biochar on the germination of corn seeds in a coppercontaminated soil was also investigated. 2. Material and methods 2.1. Soil collection, characterization and preparation The soil was collected in the 0e0.20 m layer from a fallow field at the Federal University of Sergipe experimental station, Northeast Brazil (S 10 55.77’; W 37 06.220 ). The soil was classified as Ultisol based on US Soil Taxonomy (Soil Survey Staff, 2014). Soil chemical parameters pH, EC, CEC, Ca, Mg, Al, K and P were determined. Cation exchange capacity was determined by the ammonium acetate method (Thomas, 1982); soil organic carbon by the Walkley Black method (Nelson and Sommers (1982); and soil texture by the pipette method (Day, 1965). Concentrations of extractable P, K, Ca, Mg and Cu were determined by the Mehlich 1 method (Mylavarapu et al., 2002). Soil characterization is presented in Table 1. The air-dried and sieved soil was spiked with copper sulfate (CuSO4$5H2O) to a final Cu concentration of 100 mg kg1 and wetted to 70% of its field capacity. The biochars were produced from coconut husk, orange bagasse and sewage sludge in a slow pyrolysis reactor, at 500  C. Biochar characteristics are described in Table 2. Methods used for biochar characterization are described in Gonzaga et al. (2018) (see Table 3). 2.2. Biochar incubation experiment 2.2.1. Experimental design and chemical analysis The experiment was conducted as a completely randomized design in a 3  2 factorial scheme, with three types of biochar

Table 1 Characteristics of the soil used in the study. Sand % 72

Silt 13

Clay 15

pH 4.64

EC dS m 0.63

OC 1

CEC 1

g kg 11.6

cmolc kg 1.88

Al 1

mg kg 40.5

Ca

Mg

P

K

Cu

144

79.0

1.82

25.4

0.17

1

M.I.S. Gonzaga et al. / Chemosphere 240 (2020) 124828 Table 2 Characteristics of the biochars used in the Cu-contaminated soil. Biochar characteristics

Coconut shell

Orange bagasse

Sewage sludge

Volatile matter (%) Ash (%) Fixed C (%) Cua (mg kg1) Total Cu (%) pH CEC (cmolc kg1) Surface area (m2 g1) Water retention capacity (%)

15.8 10.8 67.2 0.09 0.001 10.5 69.7 65.4 450

17.6 16.1 58.7 0.15 0.002 10.0 63.5 22.6 190

26.0 36.0 34.1 0.49 0.004 7.28 22.2 98.8 19

a

Citric acid extractable.

(coconut husk (CHB), orange bagasse (OBB) and sewage sludge (SSB)) and 2 rates of application (30 t ha1 and 60 t ha1), with four replications. A control treatment without biochar was added, with three replications. Biochar dose in g kg1 of soil was calculated according to a 0e20 cm soil layer and soil bulk density of 1.4 g kg1.

Table 3 Total Cu content and recovery rates of the sequential extraction procedure performed in the Cu-contaminated soil after 24 months laboratory incubation with coconut husk (CHB), orange bagasse (OBB) and sewage sludge (SSB) biochar. Means followed by the same letter in a column are not statistically different according to Tukey-HSD test at 5% level.for 24 months. Treatment

Total Cua (mg kg1)

Total Cu fractions (mg kg1)

Recovery ratec (%)

Control CHB30 CHB60 OBB30 OBB60 SSB30 SSB60

120.4ab 109.3abc 98.12bc 117.3bc 101.4a 119.4a 92.51c

112.3b 104.0c 111.7bc 128.5a 115.3b 115.6b 108.7bc

93.47c 96.15bc 113.8a 109.6 ab 113.8a 96.88bc 117.7a

a b c

Soil total Cu concentration was measured by the 3050 EPA method. Mean (standard deviation). The recovery rate was calculated as: (total Cu fractions/total Cu)100.

Each dose of biochar was incorporated with a 500 g air-dried soil in a 1000-mL plastic container with lids, and homogeneously mixed. Deionized water was added to reach 70% field capacity. The aging process was simulated in the laboratory through an incubation experiment. Periodically during the incubation, deionized water was cautiously sprayed to bring samples back to 70% field capacity by weighting the pots. The experiment was kept under laboratory environment with constant temperature of 25  C. One soil sample of approximately 10 g was collected from each plastic container in the first week and in the 24th month of incubation for the determination of soil pH, organic matter content and concentration of available copper. Soil pH was determined according to Gaskin et al. (2008). Soil organic matter was determined according to the Walkley-Black method (Nelson and Sommers, 1982). Available Cu was determined following extraction with 1% citric acid (Wu et al., 2004). Soil samples from the 24th month incubation were also subjected to a sequential extraction procedure, according to Salbu et al. (1998), and to a seed germination test through a Petri dish bioassay (Solaiman et al., 2012).

2.2.2. Sequential extraction procedure For the fractionation procedure, soil samples were collected from each experimental pot, air-dried and sieved to 2 mm sieve. Approximately one gram of the copper-contaminated soil treated

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with biochar was weighed into 50 mL polypropylene centrifuge tube to separate copper into six operationally defined fractions, according to Salbu et al. (1998) modified from Tessier et al. (1979), as follows: Fraction 1: Soluble þ exchangeable Cu, extracted with 15 mL of 0.1 M CaCl2; Fraction 2: Specifically adsorbed Cu, extracted with 20 mL of 1 M NH4OAc (Ammonium acetate), at pH 5; Fraction 3: Fe and Mn oxide bound Cu, extracted with 20 mL of 0.04 M NH2OH$HCl (hydroxylamine hydrochloride) in acetic acid, at pH 3; Fraction 4: Organic matter strongly bound Cu, extracted with 15 mL of 15% H2O2 at pH 2, followed by the addition of 5 mL of 3.2 M NH4OAc in 20% HNO3; Fraction 5: Residual fraction, extracted with 10 mL of 7 M HNO3. In addition, total soil Cu was measured according to USEPA Method 3050B (USEPA, 1996). The recovery rate was calculated according to the equation: Recovery rate (%) ¼ (Total Cu fractions/total Cu) x 100. Analysis of copper was performed by Flame atomic absorption spectrometry (FAAS). Analytical reagent grade chemicals and double-distilled deionized water were used for preparing all solution. One blank, one duplicate and one spiked sample were included for every 20 samples. Mobility factor (MF) of copper was calculated as the ratio between the sum of F1 and F2 and total copper concentration in all 5 fractions, as follows: MF (%) ¼ {(F1 þ F2)/(F1þF2þF3þF4þF5)} x 100. 2.2.3. Germination bioassay For the germination bioassay, 5 g of air-dried soil from each treatment were transferred to Petri dishes (8.5 cm diameter) on a layer of 41 mm filter paper moistened with 20 mL deionized water. Each Petri dish received fifteen evenly distributed corn (Zea mays L.) seed (Solaiman et al., 2012). The procedure was performed with four replications, according to the procedure described by Morrison and Morris (2000). All petri dishes were covered with lids and incubated in the dark at 25  C for 72 h. The number of germinated seeds was counted and germination percent determined. Root and cotyledon lengths were measured and reported as the sum from each dish (cm per dish). Roots and cotyledons were dried at 60  C for 48 h and weighed to determine dry mass. 2.5. Statistical analysis Analysis of variance (ANOVA) and Tukey's multiple range tests (P < 0.05) were used to determine the statistical significance of the biochar treatment effects on Cu availability and seed germination using SISVAR software package (Ferreira, 2011). Variability in the data was also expressed as the standard deviation of four replicates. 3. Results and discussion 3.1. Effect of biochar on copper availability Citric acid extractable Cu concentration varied from 67.5 to 78.8 mg kg1 (1 week incubation) and 51.8e72.0 mg kg1 (24 months incubation) (Fig. 1a). Considering that the total Cu concentration in the present study was 100 mg kg1 and that only about 1e20% of Cu in most soils is readily bioavailable (Marschner and Marschner, 2012), the concentration of available Cu observed in our study was rather high, even after 2 years of incubation. Biochar significantly (P < 0.05) affected the availability of Cu in the contaminated soil (Fig. 1a). However, its effect was dependent

Fig. 1. Concentration of citric acid extractable Cu (a), soil pH (b) and organic carbon (c) in a Cu-contaminated soil after incubation for one week and for 24 months with biochar from coconut husk (CHB), orange bagasse (OBB) and sewage sludge (SSB) at two rates of application (30 and 60 t ha1). Values are mean ± standard deviation of four replicates. Means followed by the same letter within the same period of evaluation are not statistically different according to Tukey-HSD test at 5% level.

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upon the type of biochar. One week after incubation, CHB and OBB significantly reduced Cu availability by 12.6% and 8.30%, respectively, regardless of the rate of application. Two years later, Cu availability was reduced by 18.8% (CHB30 and CHB60), 25.4% (OBB30 and OBB60) and 17.2% (SSB30 and SSB60). Compared with the control, 60 t ha1 of OBB was the most efficient treatment, which reduced Cu availability in approximately 28.0%, likely due to the highest increase in soil pH (Fig. 1 b) and soil organic carbon (Fig. 1c). The availability of Cu decreased with time in all treatments, including the control soil. Considering that it is a spiked Cucontaminated soil with low pH and low organic matter content, a high Cu solubility and availability was expected. Gonzaga et al. (2018) observed interesting results in a greenhouse study where these same types of biochar were applied to a Cu-contaminated soil to growth Indian mustard plants. In their study, the availability of copper was evaluated at 30, 50 and 70 days after biochar application. They observed that Coconut husk biochar did not influence copper availability, orange bagasse biochar reduced and sewage sludge biochar increased the availability of copper. The difference between their results and ours lies on the presence of plants, which likely influenced soil reactions, as well as in the contact time between biochar and the copper-contaminated soil. Regardless of the type of feedstock and rate of application, biochar significantly increased both soil pH (0.67e2.89 pH units) and carbon content (35e152% in the first week and 47e232% after 24 months). The increase in soil pH was observed shortly after the application of biochar to the soil and it was kept approximately constant throughout the incubation study (Fig. 1b), except for the SSB, which was more effective with time. OBB increased soil pH in 1.81 (30 t ha1) and 2.86 (60 t ha1) units, being the most efficient treatment. CHB increased soil pH in 0.82 (30 t ha1) and 1.81 (60 t ha1) unit. SSB increased soil pH in 0.96 (30 t ha1) and 1.49 (60 t ha1) unit. According to Li et al. (2016) and Zhou et al. (2017), who observed similar results regarding the effect of biochar on the increase in soil pH, biochar can also increase the soil buffering capacity and reduce heavy metal mobility. Soil organic carbon increased with time (except CHB at 30 t ha1) and rate of application in all biochar treatments. At the application rate of 60 t ha1, organic carbon was CHB > OBB > SSB two years after biochar application. At the application rate of 30 t ha1, organic carbon was OBB > SSB > CHB two years after biochar application. The biochar from sewage sludge was the least efficient in increasing the soil organic matter content, which was likely related to its lower inherent carbon content (Table 1). It is important to point out that the measured oxidizable carbon represents only a very small fraction of the carbon content in biochar and, for that reason, the addition of biochar to soil did not cause a huge increase in soil carbon in the present study. However, the evaluation of carbon after 24 months of biochar-soil incubation showed that the pool of oxidizable carbon increased in all biochar as compared with the previous measurement, which was not observed in the control soil. All three biochars could be successfully used to reduce the availability of Cu in soil, however OBB was the most effective when applied at the high rate probably due to highest increase in soil pH, which in turn increases the number of negative sites on the surface of soil colloidal particles, both mineral and organic, to bind Cu ions. The weathered Ultisol used in this study presents mineral particles such as kaolinite and Fe oxides whose variable charges depend on soil pH. However, considering the ideal range of soil pH for plant growth (5.5e6.5), CHB (60 t ha1) which also had the highest contribution in soil carbon, could be a better option than OBB at the same application rate, followed by OBB at 30 t ha1. Increasing soil pH beyond 7 could reduce the availability of P and micronutrients and could impair plant growth in the contaminated soil, reducing

5

the remediation efficiency. The use of SSB as a soil conditioner in Cu-contaminated soil was also proven efficient; however, its effect is time dependent. It needs more time in contact with soil in order to increase soil pH, oxidizable organic carbon and, therefore, to reduce Cu availability. Zhou et al. (2017) applied sewage sludge biochar to a plintic soil contaminated with heavy metals and found reduced concentration of Cu, Cd, Pb and Zn in the leachate collected from a column experiment. The authors attributed their results to some characteristics of the biochar such as large specific surface area, porous structure, and high number of surface functional groups which facilitate the formation of insoluble heavy metal compounds through chelation and complexation processes. 3.2. Effect of biochar on the distribution of Cu in soil Regardless of the treatment, the sequential extraction procedure applied to the Cu-contaminated soil after 24 months of incubation, with or without biochar, indicated that copper was mainly associated with the specifically adsorbed fraction F2 (27.6e52.1%) and strongly bound to organic matter F4 (27.6e50.7%), following by the Fe and Mn bound fraction F3 (6.04e22.7%), the residual fraction F5 (5.50e10.1%), and the soluble þ exchangeable fraction F1 (0.04e1.54%) (Fig. 2a).

Fig. 2. Copper distribution among soil fractions after 24 months laboratory incubation with biochar from coconut husk (CHB), orange bagasse (OBB) and sewage sludge (SSB) at two rates of application (30 and 60 t ha1). Values are mean ± standard deviation of four replicates.

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The low concentration of soluble and exchangeable copper confirms the highly reactive nature of Cu in soil and contradicts the high amount of Cu extracted by citric acid (Fig. 1a), which likely also accessed some of the Cu associated with other fractions, especially the specifically bound fraction. According to Kabata-Pendias (2011), copper is the least mobile trace element in soil because it adsorbs strongly to clay minerals, iron and manganese oxides, and to organic matter, forming highly stable organic complexes. McLaren and Crawford (1973) applied a similar sequential extraction procedure to a Cu-contaminated soil and observed that more than 30% was associated with organic matter, which mostly controlled Cu solubility. That is because copper ions have strong affinity for the specific sites of the organic molecules, forming inner sphere complexes with humic substances. Hence, the distribution of copper among different fractions in soil is crucial to control its concentration in the soil solution and to understand its elemental behavior in the environment, especially its bioavailability and leaching potential. In that sense, the exchangeable Cu is bioavailable while the specifically adsorbed, the strongly bound to organic matter and the Fe and Mn bound are potentially available. The residual fraction is unavailable (Yang et al., 2011). The different types and rates of application of biochar differently influenced the distribution of Cu in the contaminated soil (Fig. 2a). CHB60 significantly reduced F1 and F4 by 61.4% and 16.0%, respectively. OBB changed Cu distribution in all fractions and varied with the rate of application; OBB30 increased F1, F3, F4 and F5 by 54.5%, 24.0%, 32.2% and 64.1%, respectively; and reduced F2 by 39.8%. OBB60 reduced F1 (61.8%) and increased F5 (84.4%). SSB30 strongly reduced Cu concentration in F1 (96.2%), F2 (34.0%), and F3 (32.2%), and increased F4 (54.4%) and F5 (35.7%); SSB60 reduced F1 (57.5%) and F3 (59.3%). The reduction in the most available fraction (F1) observed in the biochar treatments is likely related to the raise in soil pH due to the liming effect of biochar. An increase in pH from 4.5 to over 6 (Fig. 1b) certainly stimulates the hydrolysis of copper ions and the formation of copper oxy(hydroxide) species, leading to a reduction in the activity and availability of the element (KabataPendias, 2011). The transfer of copper from the most available (F1 and F2) to less available form (F4) caused by some biochar treatments (OBB and SSB30) could be associated to copper retention on biochar surfaces through complexation reactions, as already observed in the study of Bakshi et al. (2014) who used Fourier-transform infrared spectra to investigate copper interaction with wood-derived biochar in two contaminated sandy soils. As biochar ages in soil, it is expected a higher degree of interaction with copper due to oxidation processes that occur on the surface of biochar, enhancing the formation of carbonyl, carboxyl, and phenolic groups that are able to complex copper ions. The oxidation of aging biochar surfaces is also responsible for the increase in biochar CEC which also contributes to the increase in copper immobilization in less available fractions (F2) (Adriano, 2001). In addition, Fe and Mn oxides are strong competitive sites for copper in contaminated soils, leading to copper precipitation and immobilization. However, in the present study, only CHB and OBB increased copper association to Fe and Mn oxides, which are reducible forms of copper. Similar results were also observed by Fang et al. (2016) who applied sewage sludge biochar to a contaminated soil. Our results are also in line with Salam et al. (2018) who reported decreased copper concentration in the most available fraction and increased copper association with less available fractions when biochars from rapeseed residues and rice straw were applied to a contaminated soil. However, we observed a broad difference among the biochars tested in the present study regarding their interaction with copper in the copper-contaminated soil, which is

probably related to the different composition of the materials in terms of other cations such as Ca, K, Mg and Zn, since they can compete for sorption sites. In addition, there are also huge differences related to the water holding capacity of the biochars (Table 1). The presence of available water can interfere with soilbiochar interactions and modify the chemical and physical reactions in the system. In order to understand copper mobility in the contaminated soil, the Mobility Factor (MF) (Fig. 2b) was determined according to Yusuf (2007). MF is a relative index that takes in consideration the two first fractions of the extraction procedures (F1 and F2) as related to the sum of all fractions. It is used to access copper mobility on the basis of the relative content of copper fractions that is weakly bound to soil components. The results showed a high mobility (33e55%) of copper in soil. All biochar reduced the MF index; however, OBB and SSB30 were the most effective. Even though the soil was spiked with copper two years before the evaluation, the artificial contamination may have played a role on the high copper mobility observed in the soil. Yusuf (2007) reported MF values lower than 10% for copper in a waste site that had been active for more than 30 years. Relatively acceptable recovery rates (93.47e117.7%) were obtained for the fractionation procedure applied in the present study (Table 2). 3.3. Effect of biochar on corn seed germination on Cu-contaminated soil High concentration of available Cu can impair seed germination and plant growth in normal crop plants. Biochar can also present detrimental effect to seed germination, especially immediately after its incorporation to soil. Therefore, we evaluated the effect of biochar on corn seed germination and seedling development in a Cu-contaminated soil after 24 months of incubation. Overall, seed germination was low and varied from 50% to 79% (Fig. 3a). Even though all biochars reduced Cu availability (Fig. 1a), only two of them (CHB30 and OBB60) improved seed germination in approximately 26%. In fact, OBB60 was the most effective in reducing Cu availability and increasing soil pH, which likely improved the germination media. The other biochar treatments reduced seed germination by approximately 20%. Gonzaga et al. (2017a) found severe inhibition in corn seeds germination in a Petri dish bioassay with sewage sludge biochar produced in a TLUD device. The authors related their results to the incomplete sludge carbonization which increased the volatile matter content of the biochar. It was expected that the long incubation time of the soilbiochar system would reduce the adverse effect of the volatile matter as well as the concentration of soluble toxic substances that might be present in the young biochar (Gonzaga et al., 2017b). In addition to the high volatile matter content, SSB also presented high ash content (Table 1) which likely increased the electrical conductivity of the medium and reduced the water absorption capacity of the seed, reducing seed germination. Even though some biochar treatments such as SSB30 and SSB60 reduced the percent germination of the corn seeds, the germinated seeds developed into vigorous plantlets which presented good cotyledon and root development (Fig. 3b, c, d and e). The presence of toxic organic compounds in the volatile matter fraction of the SSB was reported by Hale et al. (2012) and could be the cause of the reduced percent germination. However, the seeds that successfully germinated in the SSB treatments were benefited by the greater amount of nutrients in the growing media. In addition, SSB retains less amount of water than the other two biochars, which could also be related to more available water to the young plantlets. The effect of biochar was more pronounced in the root than in

M.I.S. Gonzaga et al. / Chemosphere 240 (2020) 124828

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Fig. 3. Seed germination and plantlets characteristics as affected by coconut husk (CHB), orange bagasse (OBB) and sewage sludge (SSB) biochar after 24 months laboratory incubation in a Cu-contaminated soil. Values are mean ± standard deviation of four replicates. Means followed by the same letter are not statistically different according to Tukey-HSD test at 5% level.

the shoot of the young plantlets except for CHB60, which reduced shoot mass by almost 40% (Fig. 3b) and shoot length by over 47% (Fig. 3d). Due to its great water retention capacity (Table 1), CHB60 may have affected the availability of water in the germination medium, what was probably enough for seed germination but not sufficient for plantlet development. When applied at the same rate, OBB60 and SSB60, which present lower water retention capacity (Table 1) as compared to the CHB, significantly increased root mass and length (Fig. 3c,e). Therefore, the different properties of each biochar should always be taken into consideration when applying biochar as soil amendment. Overall, CHB30 increased seed germination and shoot length in 20% and root length and root number in 86%, showing great potential in Cu-contaminated soil. The increase in seed germination is of paramount importance since it warrants stand uniformity. This result associated with a good development of the plantlets can potentially assure a better crop performance and productivity.

4. Conclusion Even though all biochar used in the present study showed potential to increase soil pH and soil carbon and reduce the availability of copper, sewage sludge biochar is probably the most interesting due to its abundance worldwide as well as the many problems faced for the proper disposal of the uncharred residue. Therefore, using sewage sludge biochar can be an effective and environmentally-friendly low-cost strategy to improve soil quality in farmland and to remediate copper-contaminated soil. However, to assure a high germination rate of the crop seeds, SSB need to be properly produced in order to reduce the volatile matter content. It is also recommended a long incubation period of the biochar with soil. Biochar from orange bagasse and sewage sludge, when applied at the rate of 30 t ha1, were the most effective in modifying the distribution of copper among soil fractions. It decreased F1 and F2

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