Biochars (BCs) are products of thermal degradation of organic materials by pyrolysis (Lehmann and Joseph, 2009). The BCs derived from agricultural biomass wastes are increasingly regarded as beneficial materials for soil and environmental remediation (Lehmann
Earthworms have been considered as the most representative soil fauna worldwide, since they perform many essential and beneficial functions in soil ecosystems, including decomposition, nutrient mineralization, and soil structure improvement (Edwards and Bohlen, 1996). Their ability to perform these functions can be inhibited upon exposure to harmful substances. Many ecotoxicological studies have used earthworms as a model system to assess the potentially toxic materials in soils (Amorim
Several studies have focused on the beneficial effects of BCs on the crop growth, reduction of greenhouse gas emission, and removal of toxic compounds (Woolf
Previous studies have shown different response of earthworms to biochar in soil. In some cases, earthworms exhibit a preference for soils amended with biochar (Chan
In order to develop appropriate recommendations for biochar application, the potential impacts on soil biota, specifically earthworms, need to be identified. The present study was undertaken to evaluate the effect of various BCs, i.e. perilla and sesame meals, and pumpkin seeds on the survival, growth, reproduction, and oxidative stress, measured by catalase (CAT) and 8‐hydroxydeoxyguanosine (8‐OHdG) activities on the earthworms.
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Biochar preparation and characterization
The BCs were produced from perilla and sesame meals, and pumpkin seed at two different temperatures (300 and 550℃) by modifying the procedure reported by Yang
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Artificial soil preparation and incubation
Artificial soil was prepared by mixing 10% Sphagnum peat moss (previously sieved through 2 mm mesh), 20% kaolin clay, and 68% quartz sand as described by US EPA guideline (US EPA, 1996). 2% calcium carbonate was added to adjust the soil pH to 6.5 ± 0.5, and moistened to 35% by weight with Milli‐Q ultrapure water. The pyrolyzed BCs at two temperatures were amended with 300 g soil at an application rate of 5%, and incubated for a week to allow equilibration. All the treatments per experiment were performed in triplicates.
In this study, the earthworm
The earthworms exposed to BC‐treated soil were picked out, and depurated on a hydrated filter paper (Whatman®, Brentford, UK).The earthworm tissue was homogenized by a moto‐homogenizer (ART‐MICCRA D‐8, Mullheim, Germany) on ice with a cold buffer (i.e. 100 mM potassium phosphate, pH 7.0, 2 mM EDTA) for CAT activity. For the measurement of 8‐ OHdG as an indicator of oxidative DNA damage, the earthworm was also homogenized with cell extraction buffer (Invitrogen, Camarillo, CA, USA), 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (Amresco, Cochran Solon, OH, USA). The homogenized samples were centrifuged at 13,000 rpm for 15 min, and the supernatants were taken for CAT and 8‐OHdG measurements at 0, 10, 20 days. The CAT activity was assayed by measuring the formaldehyde produced by the reaction of methanol with enzyme in the presence of H2O2. The amount of formaldehyde was determined using purpald as a chromogen at 595 nm. One unit of CAT was defined as the amount of enzyme to cause the formation of 1 nmol formaldehyde per min at 25℃. Oxidative DNA damage test was performed using OxiSelectTM Oxidative DNA damage ELISA Kit (Cellbiolabs, San Diego, CA, USA). Briefly, the supernatants were added to an 8‐OHdG/BSA conjugate pre‐absorbed EIA plate. After a brief incubation for 10 minutes, an anti‐8‐OHdG monoclonal antibody was added, followed by an HRP conjugated secondary antibody. Oxidative DNA damage was assayed by measuring the 8‐OHdG content at 450 nm.
All calculations and standard deviation between the replicates were done using the graphing software, SigmaPlot (version 10.0). Whether differences between the treatments were statistically significant was determined using Student’s t‐tests at the 95% confidence level.
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Characterization of biochars
Some of the physico‐chemical characteristics, i.e. pH, EC, yield, moisture, ash content, mobile, and resident matters of BCs derived from perilla, sesame, and pumpkin seeds are shown in Table 1. The pH and EC values for BC produced at 550℃ ranged from 7.81‐10.30 and 0.55‐1.06 mS/cm, respectively, which were higher than those at 300℃. The BCs produced from perilla and sesame meals at 550℃ showed high pH values over 10.0, and it reduced to 7.96 and 8.18, respectively, when amended with artificial soil at an application rate of 5%. Also, the EC values of BCs produced at 550℃ were reduced to 0.22 and 0.43 mS/cm, respectively, when amended with soil. The proportion of yield and mobile matter in the selected BCs at 550℃ were 1.2‐2.5 times lower than those at 300℃. In contrast, the ash and resident matter in BCs at 550℃ were increased to a maximum of two folds compared to those at 300℃. The moisture content at both temperatures of BCs derived from perilla, and sesame meals, and pumpkin seed were similar.
The pH, electrical conductivity, and proximate analysis of various biochars derived from perilla, and sesame meals, and pumpkin seed at 300 and 550℃.
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Trace metal(loid) concentrations
The contents of trace metal(loid)s in BCs derived from perilla, and sesame meals, and pumpkin seed are shown in Table 2. The BCs produced at 300 and 550℃ showed high contents of zinc (Zn), copper (Cu), chromium (Cr), and cadmium (Cd) in the range of 149.92‐474.52 mg/kg, 60.32‐131.60 mg/kg, 3.01‐∼12.5 mg/kg, and 0.05‐2.77 mg/kg, respectively. These contents were approximately 2‐3 times higher in BCs pyrolyzed at 550℃ than those at 300℃. No significant difference in lead (Pb) contents was observed in BCs produced at both temperatures. The arsenic (As) contents were not detected in BCs pyrolyzed at 300 and 550℃. In pumpkin seed BC, the contents of Cd were not detected and the Ni contents were two folds higher at 300℃ rather than at 550℃
The contents of trace metal(loid)s in biochars derived from perilla, and sesame meals, and pumpkin seed at 300 and 550℃
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Effect of biochar on the growth, mortality, and cocoon production of earthworms
The survival, average weight loss, and cocoon numbers in the earthworms were determined after 28 days of exposure to soil amended with 5% BC derived from perilla, and sesame meals, and pumpkin seed at 300 and 550℃ (Table 3). All the worms exposed to BC‐amended soil for 28 days survived. Average weight loss (%) of
The survival, average weight loss (%), and cocoon numbers of earthworms and soil pH after 28 days exposure to soil amended with various biochars
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Enzyme activity measurements
The CAT enzyme activity performed to assess the oxidative cellular damage of
The 8‐OHdG activity as a biomarker of oxidative DNA damage on the earthworm
This study examined the survival, growth, reproductive tests, and oxidative DNA damage tests on earthworm