Physicochemical features, metal availability and enzyme activity in heavy metal-polluted soil remediated by biochar and compost

Journal Pre-proofs Physicochemical features, metal availability and enzyme activity in heavy metal-polluted soil remediated by biochar and compost Jiayi Tang, Lihua Zhang, Jiachao Zhang, Liheng Ren, Yaoyu Zhou, Yuanyuan Zheng, Lin Luo, Yuan Yang, Hongli Huang, Anwei Chen PII: DOI: Reference:

S0048-9697(19)34742-4 https://doi.org/10.1016/j.scitotenv.2019.134751 STOTEN 134751

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Science of the Total Environment

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23 July 2019 28 September 2019 29 September 2019

Please cite this article as: J. Tang, L. Zhang, J. Zhang, L. Ren, Y. Zhou, Y. Zheng, L. Luo, Y. Yang, H. Huang, A. Chen, Physicochemical features, metal availability and enzyme activity in heavy metal-polluted soil remediated by biochar and compost, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.134751

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Physicochemical features, metal availability and enzyme activity in heavy metal-

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polluted soil remediated by biochar and compost

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Jiayi Tang, Lihua Zhang, Jiachao Zhang*, Liheng Ren, Yaoyu Zhou*, Yuanyuan Zheng, Lin

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Luo, Yuan Yang, Hongli Huang, Anwei Chen

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College of Resources and Environment, Hunan Agricultural University, Changsha 410128,

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China

7 8

*

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Agricultural University, Changsha 410128, China. Tell: +86 731 84673567; fax: +86 731

Corresponding authors. Address: College of Resources and Environment, Hunan

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84673627.

E-mail

addresses:

11

[email protected] (Y. Zhou).

[email protected]

(J.

Zhang)

and

12

Physicochemical features, metal availability and enzyme activity in heavy metal-

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polluted soil remediated by biochar and compost

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Jiayi Tang, Lihua Zhang, Jiachao Zhang*, Liheng Ren, Yaoyu Zhou*, Yuanyuan Zheng, Lin

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Luo, Yuan Yang, Hongli Huang, Anwei Chen

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College of Resources and Environment, Hunan Agricultural University, Changsha 410128,

17

China

18 19

*

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Agricultural University, Changsha 410128, China. Tell: +86 731 84673567; fax: +86 731

21

84673627.

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[email protected] (Y. Zhou).

Corresponding authors. Address: College of Resources and Environment, Hunan

E-mail

addresses:

[email protected]

1

(J.

Zhang)

and

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Abstract: Biochar and compost have been widely used for pollution remediation of heavy

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metals in soil. However, little research was conducted to explore the efficiency of biochar,

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compost and their combination to reduce heavy metals availability, and the effects of their

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additive on soil biological properties are often neglected. Therefore, this study investigated

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the effects of biochar, compost and their combination on availability of heavy metals,

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physicochemical features and enzyme activities in soil. Results showed that adding

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amendments to polluted soil significantly altered soil properties. Compared to the separate

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addition of biochar or compost, their combined application was more effective to improve

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soil pH, organic matter (OM), organic carbon (TOC) and available potassium (AK). All

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amendments significantly decreased the availability of Cd and Zn, but slightly activated As

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and Cu. In addition, soil enzyme activities were activated by compost and inhibited by

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biochar, but exhibited highly variable responses to their combinations. Pearson correlation

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analysis indicated that electrical conductivity (EC) and AK were the most important

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environmental factors affecting metal availability and soil enzyme activities including

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dehydrogenase, catalase, β-glucosidase, urease, acid and alkaline phosphatase, arylsulfatase

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except for protease and invertase. Availability of As, Cu, Cd and Zn affected dehydrogenase,

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catalase and urease activities. These results indicated that biochar, compost and their

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combination have significant effects on physicochemical features, metals availability and

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enzyme activities in heavy metal-polluted soil.

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Keywords: Heavy metal; Soil; Enzyme activity; Compost; Biochar

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1. Introduction

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Human activities, such as fertilizer application, chemical manufacturing, mining,

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smelting, tanning and fossil fuel combustion, are the main causes of heavy metal

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accumulation in soil (Beiyuan et al., 2017; Liu et al., 2019a, 2019b; Tang et al., 2019).

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Heavy metals are generally non-degradable, and their accumulation is likely to cause soil

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pollution and threaten human health (Liu et al., 2019c). A considerable number of countries

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in the world are being or have been threatened by heavy metal pollution in soil, including

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China, the United States, Italy, Mexico, etc (Tang et al., 2019). For this situation, the

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remediation of heavy metal contaminated soil has received extensive attention. A large

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number of studies have focused on immobilizing or removing heavy metals in soil with

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various organic and inorganic additives (Lu et al., 2017).

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Biochar is a carbon-rich material produced by biomass pyrolysis under oxygen-limited

55

conditions (Wang et al., 2019; Zeng et al., 2018). It has many special adsorptive properties,

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including the presence of various functional groups, large surface area, high porous structure,

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surface pH and cation exchange capacity (Nie et al., 2018; Yang et al., 2016b), thus has

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been widely used in soil bioremediation of different heavy metals (Huang et al., 2017; Yoo

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et al., 2018). Similarly, compost has the ability to reduce mobile and exchangeable metal

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fraction of contaminated soil and has been used as another highly effective amendment for

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heavy metals (Liang et al., 2017). Previous research commented that biochar and compost

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are cheap and effective additives during soil remediation, and have certain effects on each

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other's performance (Zeng et al., 2015). Biochar can affect the humification process during

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composting, and conversely, the surface of biochar can be oxidized by microbial

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communities and humus in compost (Liang et al., 2017). The interaction between above two

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amendments induces possible changes in properties of each other, which may subsequently

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affect their remediation effectiveness in soils (Beesley et al., 2014; Karami et al., 2011; 3

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Liang et al., 2017). However, there is little literature on the efficiency of biochar, compost

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and their combination to reduce the availability of heavy metals.

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The effects of biochar and compost on soil biological properties are often neglected,

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although they have proven to be attractive for the remediation of heavy metal contaminated

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soils. The ideal amendments should not only reduce the availability of potentially toxic

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metals in soil, but also improve the biological state (Garau et al., 2019). Soil enzymes play

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critical roles in organic matter decomposition, redox reactions and nutrient cycling. Their

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activities indicate the degree of biochemical reactions in soil, and can serve as important

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biological indicators for evaluating quality of soil contaminated by heavy metals (Tang et

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al., 2019). Soil amendments can directly and indirectly affect soil enzymes activity. For

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instance, Mackie et al. (2015) indicated the addition of biochar and compost altered the

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activities of invertase, phosphatase and arylsulfatase. Additionally, soil enzymes are highly

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sensitive to the changes in nutrient availability and physicochemical properties, while

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biochar (Nie et al., 2018; Yoo et al., 2018) and compost (Liang et al., 2017) are widely

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considered to have the ability to alter soil quality. Biochar (Yang et al., 2016b; Yoo et al.,

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2018) and compost (Arif et al., 2018) can improved soil available phosphorus. Biochar (Sun

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et al., 2012) and compost (Beesley et al., 2014) can significantly increase soil pH, while pH

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affects the stability and dissociation state of enzymes (Yang et al., 2016a). Similarly,

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changes in heavy metals induced by biochar (Jia et al., 2017) and compost (Garau et al.,

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2019) can also affect soil enzyme activity. However, to our knowledge, little information is

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available about the effects of biochar/compost and their combined addition on enzyme

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activities in soils contaminated by heavy metals. The relationships between enzyme

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activities and physicochemical properties, bioavailability of the heavy metals have been

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rarely evaluated simultaneously.

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Thus, the soil physicochemical and biological properties were investigated in heavy

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metal-polluted soils remediated by biochar, compost and their combination, respectively.

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The aims of this study were: (i) to determine the efficiency of biochar, compost and their

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combination to reduce the bioavailable fraction of heavy metals; (ii) to explore the effects

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of different soil amendments on enzymes activity; and (iii) to investigate the relationships

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between physicochemical factors, bioavailable fraction of heavy metals, and enzymes

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activities. This study will deepen our insight into the remediation efficiency for heavy metal

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pollution and the microbiological mechanism of different remediation strategies in soils

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polluted by heavy metals.

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2. Materials and methods

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2.1. Soil samples and amendments characterization

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Soil samples were collected from Changde City, Hunan Province, China. Soil in this

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area was polluted by heavy metals (e.g., Cd, Zn, As, and Cu) because of mining production

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and agricultural activities. Soil samples were taken from the topsoil (0-20 cm). After picking

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up the gravels, animal and plant residues, the soil was placed in sterile sealed bags and

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brought back to laboratory. In order to mix thoroughly the soil and additives, the soil was

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air-dried for 1 week at room temperature, then mixed uniformly, and screened through a 2

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mm sieve. The soil was slight acidic with pH, electrical conductivity, organic matter was

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5.98 ± 0.01, 0.21 ± 0.01 ds.m-1, and 62.33 ± 4.47 g kg-1, respectively (Table 1). Biochar was

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obtained from rice straw using the tubular carbonization furnace in hypoxia condition

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(500 °C, 3 h) (Li et al., 2019; Zeng et al., 2018) and ground to pass through 10-mesh (2.00

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mm) sieve before using. Compost samples were prepared using agricultural waste (rice

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straw, vegetable leaves, et al.) according to previous studies (Ren et al., 2018; Zeng et al.,

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2011). The length of compost samples was about 0.50~1.00 cm after composting (Ren et

5

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al., 2018). The main physicochemical parameters of soil, biochar and compost were shown

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in Table 1.

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2.2. Experimental design and sample collection

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Four treatments were conducted as follows: Treatment S without any addition (control),

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Treatment S + B added with biochar, Treatment S + C added with fresh compost, and

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Treatment S + B + C added with biochar and fresh compost. Each treatment was set up with

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three replicates. The soil was mixed with biochar and compost in the following proportions:

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S: 5 kg of soil per pot.

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S + B: 5 kg of soil and 0.25 kg of biochar per pot.

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S + C: 5 kg of soil and 0.25 kg of compost per pot.

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S + B + C: 5 kg of soil, 0.25 kg of biochar and 0.25 kg of compost per pot.

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The four treatments were cultured in an artificial climate chamber with moisture

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content adjusted to ~70% at room temperature for 30 days. Subsamples were collected on

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days 0, 15, 30, respectively. Samples for enzyme activity analysis and physicochemical

130

properties determination were stored at -20oC and 4oC, respectively.

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2.3. Physicochemical property determination

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Subsamples were air-dried and passed through a 2-mm sieve before physicochemical

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measurements. The organic matter (OM), organic carbon (TOC), ammonium (NH4+-N),

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nitrate (NO3–-N), available phosphorus (AP), total potassium (TK), available potassium

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(AK), electrical conductivity (EC), pH and moisture content were determined. The moisture

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content was measured by drying samples at 105 oC for 24 h (Zhang et al., 2011). The pH

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and EC were measured in a 1: 5 (w/v) aqueous suspension (Arif et al., 2018; Liu et al.,

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2019d). The NH4+-N and NO3–-N were extracted by 2 M KCl and then measured by flow-

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injection analyzer (Zeng et al., 2011). The OM content was analyzed by dry combustion,

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and the TOC content was equal to OM/1.724 content (Zhang et al., 2011). The AP was 6

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extracted by NaHCO3 (pH 8.5)-colorimetric method (Arif et al., 2018). The AK was

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measured by NH4OAc extraction-flame photometer method (Yoo et al., 2018). The total As,

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Cd, Cu, Zn and K contents were analyzed by ICP-MS (PerkinElmer, NexION 300×, USA)

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after digested with HNO3-HF-HClO4 (Yang et al., 2016b).

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2.4. Heavy metals availability determination

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The availability after CaCl2 extraction of contaminant was usually regarded as an

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effective index of metal availability in polluted soils (Liang et al., 2017). The available

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metals (As, Cd, Cu and Zn) in differently treated soils were extracted by CaCl2 according

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to Liang et al. (2017) and were analyzed by ICP-MS (PerkinElmer, NexION 300×, USA).

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The detection limits of Cd, Zn, As, and Cu in ICP-MS were 0.005 μg L-1, 0.06 μg L-1, 0.02

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μg L-1 and 0.02 μg L-1, respectively.

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2.5. Measurement of enzyme activity

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Fresh soil subsamples were used to measure enzyme activities. The nine enzymes

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analyzed were 2 oxidoreductases (dehydrogenase, catalase), 2 C-cycling enzymes (β-

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glucosidase, invertase), 2 N-cycling enzymes (urease, protease), 2 P-cycling enzymes (acid

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and alkaline phosphatase), and 1 S-cycling enzyme (arylsulfatase). The activities of

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dehydrogenase, catalase, invertase, urease and protease were assayed on the basis of the

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production of triphenyl formazan, H2O, glucose, NH3-N and tyrosine, respectively. The

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activities of β-glucosidase, acid phosphatase, alkaline phosphatase and arylsulfatase were

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determined by the release of p-nitrophenol (PNP). The above enzyme activities

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measurement have been summarized in Table 2.

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2.6. Data analysis

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Soil physicochemical characteristics, heavy metal availability, and enzyme activity of

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four treated samples were analyzed using SPSS software (version 22). One-way analysis of

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variance (ANOVA) was used to analyze the difference of the above-mentioned parameters 7

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between different treatments. Correlations between parameters (soil physicochemical

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parameters, heavy metal availability and enzyme activity) were determined by the

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coefficients of Pearson’s correlation analysis.

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3. Results and discussion

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3.1. Effects of amendments on soil physicochemical properties

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Generally, EC, pH, OM, TOC, NO3–-N, AP, and AK were significantly changed by the

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addition of biochar and compost (Fig. 1). The highest pH value was observed in biochar-

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compost combination, while the increase of pH in biochar and compost was similar (Fig.

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1a). Previous studies showed that biochar (Chen et al., 2013; Ibrahim et al., 2016; Liang et

175

al., 2017), compost (Clemente and Bernal, 2006) and biochar-compost combination

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(Beesley et al., 2014; Liang et al., 2017) increased soil pH. However, a decrease in soil pH

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was also observed in some studies (Zeng et al., 2015). The EC increased significantly under

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the compost and biochar-compost combination addition, while it decreased slightly under

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the biochar treatment except for a slight increase on day 0 (Fig. 1b). Our discoveries were

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in contradiction with a previous study by Igalavithana et al. (2017) that indicated biochar

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improved the soil EC. Compared to unamended soil, each amendment increased the content

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of OM, TOC, AP and AK. For OM and TOC, their content in the biochar-compost

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combination treatment was the highest, then followed by biochar and compost (Fig. 1c, d).

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A considerable number of studies also found that biochar (Abujabhah et al., 2016; Chen et

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al., 2013), compost (Arif et al., 2018; Gusiatin and Kulikowska, 2016) and their combination

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(Liang et al., 2017) had a positive impact on TOC. Biochar rather than compost slightly

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increased the content of NH4+-N during the 30-day culture period, while biochar-compost

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combination reduced NH4+-N content on day 0 and then slightly increased its content on

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days 15 and 30 (Fig. 1e). Compared with the control treatment, biochar slightly reduced

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NO3–-N content, while compost and biochar plus compost significantly increased it except 8

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for slightly decreasing the content on day 0 (Fig. 1f). Similar with our findings, previous

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study also demonstrated that biochar significantly reduced the NO3–-N and slightly

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increased NH4+-N (Chen et al., 2013). For AP and AK, their content increased significantly

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under compost and biochar-compost combination treatment, but to a lesser degree in biochar

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treatment (Fig. 1g, h). Similar to our results, Yang et al. (2016b) manifested biochar addition

196

improved the AP concentration, and Arif et al. (2018) indicated composted industrial sludge

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significantly improved soil AP and AK.

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The improvements in soil performance after application of amendments may be a

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direct contribution of materials or an interaction between physicochemical properties. For

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example, the sharp drop in soil pH at the beginning of biochar or compost application might

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be related to the easily degradable OM in the materials, because organic acids could be

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released during the decomposition of organic matter (Zeng et al., 2015). Humic acid isolated

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from compost might also contribute to the reduction of pH (Zeng et al., 2015). The increase

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of pH in the compost-biochar combination was significantly higher than the addition of

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biochar or compost, which might be the result of interactions between biochar and compost.

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On the one hand, the negatively charged functional groups including phenolic, hydroxyl and

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carboxyl groups on the surface of biochar, will combine with the H+ ions in soil, thus help

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to increase the soil pH (Gul et al., 2015), while organic matter and microbe in the compost

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can adsorb to the surface and pores of the biochar to promote the formation of functional

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groups in the biochar (Liang et al., 2017). Biochar can enhance the compost humification

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process and quality by selectively adsorbing organic matter on the surface and pores of

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biochar to create a favorable environment for growth and proliferation of microorganisms

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(Liang et al., 2017). Furthermore, the correlations between soil physicochemical properties

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(Table 3) in this study indicated the content of NO3–-N and NH4+-N also affected pH, which

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were manifested by the increase in NO3–-N content led to an increase in pH, while the 9

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increase in NH4+-N content result in soil acidification. NH4+-N was proven to reaction after

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it was applied to the soil to produce H+ (Matsuyama et al., 2005), while NO3–-N may react

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with H+ to eliminate soil acidification. And the solubilization of ammonia may also result

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in an increase in pH (Gil et al., 2008). For EC, the slight decrease in biochar treatment and

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the significant increase in compost and biochar plus compost treatment might be due to the

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EC of the biochar and compost used in the experiment were 0.16 and 7.99 ds m-1,

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respectively, while that of the original soil was 0.21 ds m-1. According to Table 3, increased

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nutrient content such as AP and AK might also led to an increase in EC. Previous studies

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demonstrated that the reduction in soil EC reduction might be associated with microbial

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assimilation of NO3- and SO42- by OM decomposition (Arif et al., 2018). For TOC, biochar

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has been reported to have an organic carbon content of up to 90%, relying on the raw

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materials (Beesley et al., 2010). Compost is rich in humus substances that the main organic

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carbon reservoir for the carbon cycle (Gusiatin and Kulikowska, 2016). The increase of

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TOC might indicate the presence of organic compounds that are less difficult to degrade in

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compost (Arif et al., 2018). In addition, the literature demonstrated that biochar can improve

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TOC content by promoting the polymerization of small organic molecules through surface

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catalytic activity after adsorbing soil organic molecules (Song et al., 2019).

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3.2. Effects of amendments on availability of heavy metals

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The extractable contents of As, Cu, Zn, and Cd in different treatments were shown in

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Fig. 2. These results indicated that all amendments significantly passivated Cd and Zn, with

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the biochar-compost combination achieving the highest reduction rate, then followed by

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compost and biochar (Fig. 2a, b). Comparing with the control treatment on day 30, the

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contents of Cd and Zn were decreased by 87.1 % and 86.4 % (biochar + compost), 69.6 %

239

and 76.5 % (compost), 65.8 % and 59.9 % (biochar), respectively. However, the availability

240

of As was significantly increased by up to 374.3 %, 258.0 % and 83.3 % in biochar-compost 10

241

combination, compost and biochar treatments, respectively (Fig. 2c). Cu was also increased

242

by compost and biochar-compost combination, but it was slightly reduced by biochar

243

addition (Fig. 2d). Interestingly, the available Cu in biochar-compost treatment gradually

244

decreased over time, but this phenomenon did not occur under compost treatment. These

245

results were similar to many previous reports. For example, Beesley et al. (2010) found that

246

after contaminated soil was treated with both biochar and greenwaste compost, the

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concentrations of Cd and Zn decreased significantly, while the concentrations of labile As

248

and Cu increased by more than 30 times. Ibrahim et al. (2016) indicated that the content of

249

available Cr, Cd, Pb and Zn in the soil added with rice husk biochar decreased, but the

250

available As concentration significantly increased by 72 %. Our results were also partially

251

in contradiction with Gusiatin and Kulikowska (2016) that sewage sludge compost reduced

252

the available Cd, Ni and Zn concentration, but had no effect on the availability of Pb and

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Cu.

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The inhibition or activation of heavy metals may be partly attributed to the direct effect

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of amendments in soil. Biochar, especially with large surface area, functional groups and

256

high pH, can facilitate the immobilization of metal cations via electrostatic interactions and

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chelation between surface functional groups of biochar and heavy metals (Jia et al., 2017).

258

Surely, the surface functional component of biochar, such as negatively charged functional

259

groups, may also limit the adsorption of heavy metals such as As, thereby increasing the

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availability of heavy metals in the soil (Ibrahim et al., 2016). Compost contains a large

261

amount of humic substances, which can form stable organometallic complexes with metal

262

ions in the soil to reduce the mobility of metals (Arif et al., 2018; Gusiatin and Kulikowska,

263

2016). And the nature and performances of humic acids determine the stability constant of

264

complexes (Clemente and Bernal, 2006). Moreover, compost with a low carbon to nitrogen

265

ratio and a high proportion of humic substances to TOC can more effectively reduce the 11

266

mobility of heavy metals in soil (Gusiatin and Kulikowska, 2016). Conversely, some heavy

267

metals such as Cu may be activated by humic acid (Zeng et al., 2015). The increase in the

268

availability of As and Cu may also be introduced by materials. Biochar and compost contain

269

different contents of As and Cu (Table 1), which may explain the availability of As and Cu

270

especially under compost and combination treatment have significantly exceeded the

271

control on day 0. Additionally, soil physicochemical properties altered by soil additives may

272

indirectly affected the availability of heavy metals. Pearson correlation analysis in this study

273

showed that the CaCl2-extractable As and Cu were positively related to soil EC, AP and

274

AK, while Cd and Zn were negatively correlated with soil EC, AP, AK and OM (Table 3).

275

The increase in AP result in a decrease in the availability of heavy metals possibly by

276

precipitation and complexation with phosphate (Ahmad et al., 2012). Phosphate is

277

chemically similar to arsenate, the increased AP content caused As to be released from the

278

soil (Beesley et al., 2014). Soil OM could act as an important adsorbent for heavy metals

279

(i.e., Cd and Zn) because it contains various important functional groups, such as -COOH

280

and -OH, and these functional groups can easily bind metal ions and form strong anti-

281

desorption complexes (Chapman et al., 2013; Guo et al., 2006; Yang et al., 2016a). The

282

adsorption of OM on metals was affected by ionic strength (Chapman et al., 2013).

283

Moreover, the OM transformations such as hydrolysis, oxidation and depolymerization

284

affected the solubility of metals (Gusiatin and Kulikowska, 2016). Interestingly, pH,

285

generally considered to be one of the most important factors affecting soil metal availability

286

(Jia et al., 2017; Liang et al., 2017; Karami et al., 2011; Lu et al., 2017), in this study, the

287

effect of pH was not significant (Table 3), which may be due to pH dependence being

288

overshadowed by the presence of OM (Chapman et al., 2013).

289

3.3. Effects of amendments on enzyme activity

12

290

Dehydrogenase, catalase, β-glucosidase, invertase, urease, protease, acid phosphatase,

291

alkaline phosphatase and arylsulfatase were measured to characterize the dynamic changes

292

of microbial activities induced by soil additives (Fig. 3). Biochar treatment inhibited all

293

enzyme activities except for urease throughout the incubation period compared to the

294

control treatment. On the contrary, compost addition showed a significant promotion of all

295

selected enzyme activities, except for invertase and protease on day 0. Interestingly, on day

296

15, the activities of invertase and protease increased at an alarming rate by 742.3 % and

297

1269.9 %, respectively. Biochar-compost combination produced a highly variable effects

298

on enzyme activities, from activation to inhibition. Compared with the control, the treatment

299

promoted the activities of dehydrogenase, catalase, invertase, urease, protease and

300

arylsulfatase, except that invertase and protease activities were significantly inhibited on the

301

0th day, conversely, the activities of β-glucosidase, acid phosphatase and alkaline

302

phosphatase gradually decreased over time during the culture period, with the activities

303

were lower than that of the additive-free soil on day 30.

304

Dehydrogenase and catalase are oxidoreductases that directly alter ion valence and

305

participate in the detoxification of heavy metals (Yang et al., 2016a). Hydrolases β-

306

glucosidase, invertase, urease, protease, acid and alkaline phosphatase and arylsulfatase

307

participate in soil nutrients recycle such as C, N, P and S (Yang et al., 2016a). The reduction

308

of soil enzyme activities caused by biochar may be attributed to various reasons: (i) biochar

309

addition directly harms microorganisms, which affect enzymes production (Huang et al.,

310

2017). (ii) Biochar has the ability to adsorb a variety of organic and inorganic molecules,

311

and can inhibit certain enzymes or enzyme-substrates via adsorption or by blocking the

312

reaction sites (Elzobair et al., 2016). Moreover, high specific surface area and porous

313

materials might make substrates unusable for slowing degradation (Chen et al., 2013).

314

Conversely, the increase in enzyme activities induced by compost were likely associated 13

315

with increased substrate availability (Mackie et al., 2015) and microbial population, while

316

the highly variability in soil enzyme activities in combinations treatment may be linked to

317

the interaction between biochar and compost. However, there are also some studies showed

318

that biochar activated soil enzyme activities such as urease and invertase (Jia et al., 2017),

319

dehydrogenase and alkaline phosphatase (Chen et al., 2013). And compost reduced soil

320

enzyme activities such as urease (Abujabhah et al., 2016), acid phosphatase and urease in

321

surface soil (Arif et al., 2018). The contradictory effect of additives on soil enzyme activities

322

is partly due to the different feedstocks of remediation materials. For example,

323

Bhattacharyya et al. (2005) and Garau et al. (2019) both indicated that municipal solid waste

324

compost increased urease activity. Huang et al. (2017) used the same ratio of rice straw

325

biochar as additive, showed that alkaline phosphatase, invertase and urease activities were

326

inhibited throughout the 30-day culture period except for a slight increase in invertase and

327

urease activities on day 7. However, another study (Yang et al., 2016b) found that urease,

328

catalase and acid phosphatase activities in soil increased to varying degrees after the

329

addition of the same proportion of rice straw biochar as this experiment. The reason may be

330

that in addition to material sources, production methods, soil properties (Bailey et al., 2011),

331

amendments content (Huang et al., 2017) and enzyme activities monitored at different times

332

may also result in different responses of soil enzyme activity to additives to a large extent.

333

3.4. Enzyme activity correlation matrix

334

A considerable number of previous reports have observed that soil enzyme activity was

335

affected by heavy metals (Jia et al., 2017). Changes in soil enzyme activities may be partly

336

a response to the alterations of heavy metal availability caused by soil amendments. The

337

Pearson correlation analysis was used to analyze the relationship between soil enzyme

338

activity and heavy metals availability in this study (Table 4). The activity of dehydrogenase,

339

catalase and urease were negatively correlated with Cd and Zn (P < 0.05). Significant 14

340

positive relationships were discovered between the activity of dehydrogenase, catalase, β-

341

glucosidase, urease, alkaline phosphatase, arylsulfatase with As, Cu. Acid phosphatase

342

activity was positively related to only Cu (P < 0.01), while invertase and protease activities

343

shared no relationship with all heavy metals (P > 0.05). The increases in Cd and Zn

344

availability led to the inhibition of soil enzyme activities may be due to metal ions react

345

with enzymes sulfhydryl group, or chelate with substrates or react with enzyme-substrates

346

(Hu et al., 2014). On the contrary, the increased availability of As and Cu contributes to the

347

activation of soil enzyme activities probably because enzyme as a protein requires a certain

348

amount of heavy metal ions as a cofactor, while heavy metals can promote the coordination

349

between the enzyme active site and the substrate. Consistent with our results, many

350

researchers' conclusions also indicated catalase, urease (Yang et al., 2016b) and

351

dehydrogenase activities (Hu et al., 2014; Liang et al., 2014) were related to Cu, Zn and Cd,

352

acid phosphatase activity had no correlation with Cd, Pb and Zn (Hu et al., 2014; Yang et

353

al., 2016b), invertase and protease activities had no relationship with Cd, Cu, Pb and Zn

354

(Yang et al., 2016a). However, our discoveries also in contradiction with some studies, such

355

as the relationship between dehydrogenase with As, urease activity with As, Cd (Xian et al.,

356

2015), alkaline phosphatase with Zn (Huang et al., 2017), alkaline phosphatase,

357

arylsulfatase with Cd, Zn (Liang et al., 2014). The selected soil enzyme activities have

358

similar or different correlations with As, Cu, Cd, Zn in many studies, which may be due to

359

the different pollution levels, methods for enzyme activity measurements, soil properties,

360

etc (Yang et al., 2016a).

361

The addition of biochar, compost and biochar plus compost significantly improved soil

362

properties, which might in turn affect soil enzyme activities. Experts previously pointed out

363

that soil enzyme activities were less affected by soil physicochemical properties (Hu et al.,

364

2014), but many scholars held the opposite opinion (Chen et al., 2014; Xian et al., 2015). 15

365

Xu et al. (2015) suggested that the best indicator for predicting soil enzyme activities was

366

nutrient levels. The relationship between soil enzyme activities with physicochemical

367

properties was revealed by Pearson correlation analysis in Table 4. This analysis indicated

368

that soil EC and AK were the most important environmental factors for all selected enzymes

369

except for invertase and protease, and AP affected dehydrogenase, catalase, urease and

370

arylsulfatase activities (P < 0.01), while pH, OM, TOC, NH4+-N and NO3–-N were

371

independent of all soil enzyme activities (P > 0.05). AP and AK are not only key nutrients

372

for soil plants growth and environmental sustainability, but also a reliable symptom of soil

373

productivity (Arif et al., 2018; Yang et al. 2016a). The significant positive relationship

374

between soil enzyme activities and nutrients contents especially AK in this study may

375

support previous view that availability and quality of soil nutrients affected enzyme

376

activities, and low nutrient levels inhibited the production of soil enzyme (Xu et al., 2015).

377

Soil nutrients such as AP and AK supplied from additives may alleviate the nutrient

378

limitation of microbial metabolism and therefore enhanced the metabolic activities of

379

microbes, especially enzymes excretion. Similarly, the increase in EC induced by additives

380

significantly activated most of soil enzyme activities may indicate a positive effect of EC

381

on enzyme activities. Soil enzymes are extremely sensitive to environmental changes and

382

can serve as an excellent indicator of soil quality. However, regrettably, the response of soil

383

enzymes to soil parameters has not yet reached a consensus conclusion due to the complex

384

environmental conditions and soil types (Tang et al., 2019). There are many differences in

385

the existing literature on the relationship between soil physicochemical parameters and soil

386

enzymes. For example, Huang et al. (2017) proved that pH was negatively correlated with

387

invertase and alkaline phosphatase activities, and positively related to urease activity, but

388

Yang et al. (2016a) found that these enzyme activities were independent of pH, and Bera et

389

al. (2016) showed that a positive correlation between pH and alkaline phosphatase activity. 16

390

Xu et al. (2015) revealed the NO3–-N content activated β-glucosidase activity, while Yang

391

et al. (2016a) manifested NO3–-N had no relationship with β-glucosidase. Overall, the

392

impact of soil properties on enzyme activities of heavy metal-contaminated soils is still the

393

direction of future research, especially in the presence of additives. In addition, soil

394

microbial abundance and community changes under additives also need to be studied,

395

because soil nutrient cycling is affected by various microorganisms, and nutrients and

396

microorganisms affect soil enzyme activities.

397

4. Conclusions

398

Addition of biochar, compost and their combination to heavy metal polluted soil

399

changed physicochemical properties. The combined addition of biochar and compost was

400

more suitable as remediation agent to improve soil pH, OM, TOC and AK. All amendments

401

significantly decreased the availability of Cd and Zn, but slightly activated As and Cu. The

402

availability of As, Cu, Cd and Zn were significantly related to soil EC, AP and AK. Enzyme

403

activities were almost completely inhibited by biochar, and activated by compost. EC and

404

AK in soil were the most important factors affecting enzyme activities. Availability of As,

405

Cu, Cd and Zn affected dehydrogenase, catalase and urease activities.

406

Acknowledgements：

407

This work was jointly supported by the Hunan Key Scientific Research Project (Grant No.

408

2019WK2031, 2017SK2351), the National Natural Science Foundation of China

409

(51408219), the China Postdoctoral Science Foundation (Grant No. 2018M630901), the

410

Hong Kong Scholars Program (XJ2018029).

411

References:

412

Abujabhah, I.S., Bound, S.A., Doyle, R., Bowman, J.P., 2016. Effects of biochar and

413

compost amendments on soil physico-chemical properties and the total community

414

within a temperate agricultural soil. Appl. Soil Ecol. 98, 243-253. 17

415

Ahmad, M., Lee, S.S., Yang, J.E., Ro, H.-M., Lee, Y.H., Ok, Y.S.J.E., Safety, E., 2012.

416

Effects of soil dilution and amendments (mussel shell, cow bone, and biochar) on Pb

417

availability and phytotoxicity in military shooting range soil. Ecotoxicol. Environ. Saf.

418

79, 225-231.

419

Arif, M.S., Riaz, M., Shahzad, S.M., Yasmeen, T., Ashraf, M., Siddique, M., Mubarik, M.S.,

420

Bragazza, L., Buttler, A., 2018. Fresh and composted industrial sludge restore soil

421

functions in surface soil of degraded agricultural land. Sci. Total Environ. 619, 517-

422

527.

423

Bailey, V.L., Fansler, S.J., Smith, J.L., Bolton, Jr. H., 2011. Reconciling apparent variability

424

in effects of biochar amendment on soil enzyme activities by assay optimization. Soil

425

Biol. Biochem. 43, 296-301.

426

Beesley, L., Inneh, O.S., Norton, G.J., Moreno-Jimenez, E., Pardo, T., Clemente, R.,

427

Dawson, J.J., 2014. Assessing the influence of compost and biochar amendments on

428

the mobility and toxicity of metals and arsenic in a naturally contaminated mine soil.

429

Environ. Pollut. 186, 195-202.

430

Beesley, L., Moreno-Jiménez, E., Gomez-Eyles, J.L., 2010. Effects of biochar and

431

greenwaste compost amendments on mobility, bioavailability and toxicity of inorganic

432

and organic contaminants in a multi-element polluted soil. Environ. Pollut. 158, 2282-

433

2287.

434

Beiyuan, J., Li, J.S., Tsang, D.C., Wang, L., Poon, C.S., Li, X.D., Fendorf, S., 2017. Fate

435

of arsenic before and after chemical-enhanced washing of an arsenic-containing soil in

436

Hong Kong. Sci. Total Environ. 599, 679-688.

437

Bera, T., Collins, H.P., Alva, A.K., Purakayastha, T.J., Patra, A.K., 2016. Biochar and

438

manure effluent effects on soil biochemical properties under corn production. Appl.

439

Soil Ecol. 107, 360-367. 18

440

Bhattacharyya, P., Chakrabarti, K., Chakraborty, A., 2005. Microbial biomass and enzyme

441

activities in submerged rice soil amended with municipal solid waste compost and

442

decomposed cow manure. Chemosphere. 60, 310-318.

443

Chapman, E.E.V., Dave, G., Murimboh, J.D., 2013. A review of metal (Pb and Zn) sensitive

444

and pH tolerant bioassay organisms for risk screening of metal-contaminated acidic

445

soils. Environ. Pollut. 179, 326-342.

446

Chen, J., He, F., Zhang, X., Sun, X., Zheng, J., Zheng, J., 2014. Heavy metal pollution

447

decreases microbial abundance, diversity and activity within particle-size fractions of

448

a paddy soil. FEMS Microbiol. Ecol. 87, 164-181.

449

Chen, J., Liu, X., Zheng, J., Zhang, B., Lu, H., Chi, Z., Pan, G., Li, L., Zheng, J., Zhang, X.,

450

2013. Biochar soil amendment increased bacterial but decreased fungal gene

451

abundance with shifts in community structure in a slightly acid rice paddy from

452

Southwest China. Appl. Soil Ecol. 71, 33-44.

453

Clemente, R., Bernal, M.P., 2006. Fractionation of heavy metals and distribution of organic

454

carbon in two contaminated soils amended with humic acids. Chemosphere, 64, 1264-

455

1273.

456

Elzobair, K.A., Stromberger, M.E., Ippolito, J.A., Lentz, R.D., 2016. Contrasting effects of

457

biochar versus manure on soil microbial communities and enzyme activities in an

458

Aridisol. Chemosphere, 142, 145-152.

459 460

Frankeberger, W.T., Johanson, J.B., 1983. Method of measuring invertase activity in soils. Plant Soil. 74, 301-311.

461

Garau, G., Porceddu, A., Sanna, M., Silvetti, M., Castaldi, P., 2019. Municipal solid wastes

462

as a resource for environmental recovery: Impact of water treatment residuals and

463

compost on the microbial and biochemical features of As and trace metal-polluted soils.

464

Ecotoxicol. Environ. Saf. 174, 445-454. 19

465

Gil, M.V., Carballo, M.T., Calvo, L.F., 2008. Fertilization of maize with compost from

466

cattle manure supplemented with additional mineral nutrients. Waste Manage. 28,

467

1432-1440.

468

Gul, S., Whalen, J.K., Thomas, B.W., Sachdeva, V., Deng, H.Y., 2015. Physico-chemical

469

properties and microbial responses in biochar-amended soils: mechanisms and future

470

directions. Agr. Ecosyst. Environ. 206, 46-59.

471

Guo, X., Zhang, S., Shan, X.Q., Luo, L., Pei, Z., Zhu, Y.G., Liu, T., Xie, Y.N., Gault, A.,

472

2006. Characterization of Pb, Cu, and Cd adsorption on particulate organic matter in

473

soil. Environ. Toxicol. Chem., 25, 2366-2373.

474 475

Gusiatin, Z.M., Kulikowska, D., 2016. Behaviors of heavy metals (Cd, Cu, Ni, Pb and Zn) in soil amended with composts. Environ. Technol. 37, 2337-2347.

476

Hu, X.F., Jiang, Y., Shu, Y., Hu, X., Liu, L., Luo, F., 2014. Effects of mining wastewater

477

discharges on heavy metal pollution and soil enzyme activity of the paddy fields. J.

478

Geochem. Explor. 147, 139-150.

479

Huang, D., Liu, L., Zeng, G., Xu, P., Huang, C., Deng, L., Wang, R., Wan, J., 2017. The

480

effects of rice straw biochar on indigenous microbial community and enzymes activity

481

in heavy metal-contaminated sediment. Chemosphere, 174, 545-553.

482

Ibrahim, M., Khan, S., Hao, X., Li, G., 2016. Biochar effects on metal bioaccumulation and

483

arsenic speciation in alfalfa (Medicago sativa L.) grown in contaminated soil. Int. J.

484

Environ. Sci. Tech. 13, 2467-2474.

485

Igalavithana, A.D., Lee, S.E., Lee, Y.H., Tsang, D.C., Rinklebe, J., Kwon, E.E., Ok, Y.S.,

486

2017. Heavy metal immobilization and microbial community abundance by vegetable

487

waste and pine cone biochar of agricultural soils. Chemosphere, 174, 593-603.

488 489

Jia, W., Wang, B., Wang, C., Sun, H., 2017. Tourmaline and biochar for the remediation of acid soil polluted with heavy metals. J. Environ. Chem. Eng. 5, 2107-2114. 20

490 491

Johnson, J.L., Temple, K.L., 1964. Some variables affecting the measurement of “catalase activity” in soil1. Soil Sci. Soc. Am. J. 28, 207-209.

492

Karami, N., Clemente, R., Moreno-Jiménez, E., Lepp, N.W., Beesley, L., 2011. Efficiency

493

of green waste compost and biochar soil amendments for reducing lead and copper

494

mobility and uptake to ryegrass. J. Hazard. Mater. 191, 41-48.

495

Li, M., Ren, L., Zhang, J., Luo, L., Qin, P., Zhou, Y., Huang, C., Tang, J., Huang, H., Chen,

496

A., 2019. Population characteristics and influential factors of nitrogen cycling

497

functional genes in heavy metal contaminated soil remediated by biochar and compost.

498

Sci. Total Environ. 651, 2166-2174.

499

Liang, J., Yang, Z., Tang, L., Zeng, G., Yu, M., Li, X., Wu, H., Qian, Y., Li, X., Luo, Y.,

500

2017. Changes in heavy metal mobility and availability from contaminated wetland

501

soil remediated with combined biochar-compost. Chemosphere, 181, 281-288.

502

Liang, Q., Gao, R., Xi, B., Zhang, Y., Zhang, H., 2014. Long-term effects of irrigation using

503

water from the river receiving treated industrial wastewater on soil organic carbon

504

fractions and enzyme activities. Agr. Water Manage. 135, 100-108.

505

Liu, J., Li, N., Zhang, W., Wei, X., Tsang, D. C., Sun, Y., Luo, X., Bao, Z., Zheng, W.,

506

Wang, J., Xu, G., Hou, L., Chen, Y., Feng, Y., 2019a. Thallium contamination in

507

farmlands and common vegetables in a pyrite mining city and potential health

508

risks. Environ. Pollut. 248, 906-915.

509

Liu, J., Luo, X., Sun, Y., Tsang, D. C., Qi, J., Zhang, W., Li, N., Yin, M., Wang, J., Lippold,

510

H., Chen, Y., Sheng, G., 2019b. Thallium pollution in China and removal technologies

511

for waters: A review. Environ. Int. 126, 771-790.

512

Liu, J., Yin, M., Luo, X., Xiao, T., Wu, Z., Li, N., Wang, J., Zhang, W., Lippold, H.,

513

Belshaw, N., Feng, Y., Chen, Y., 2019c. The mobility of thallium in sediments and

514

source apportionment by lead isotopes. Chemosphere. 219, 864-874. 21

515

Liu, J., Yin, M., Zhang, W., Tsang, D. C., Wei, X., Zhou, Y., Xiao, T., Wang, T., Dong, X.,

516

Sun, Y., Chen, Y., Li, H., Hou, L., 2019d. Response of microbial communities and

517

interactions to thallium in contaminated sediments near a pyrite mining area. Environ.

518

Pollut. 248, 916-928.

519

Lu, K., Yang, X., Gielen, G., Bolan, N., Ok, Y.S., Niazi, N.K., Xu, S., Yuan, G., Chen, X.,

520

Zhang, X., 2017. Effect of bamboo and rice straw biochars on the mobility and

521

redistribution of heavy metals (Cd, Cu, Pb and Zn) in contaminated soil. J. Environ.

522

Manage. 186, 285-292.

523

Mackie, K.A., Marhan, S., Ditterich, F., Schmidt, H.P., Kandeler, E., 2015. The effects of

524

biochar and compost amendments on copper immobilization and soil microorganisms

525

in a temperate vineyard. Agr. Ecosyst. Environ. 201, 58-69.

526

Matsuyama, N., Saigusa, M., Sakaiya, E., Tamakawa, K., Oyamada, Z., Kudo, K., 2005.

527

Acidification and soil productivity of allophanic Andosols affected by heavy

528

application of fertilizers. Soil. Sci. Plant. Nutr. 51, 117-123.

529

Nie, C., Yang, X., Niazi, N.K., Xu, X., Wen, Y., Rinklebe, J., Ok, Y.S., Xu, S., Wang, H.,

530

2018. Impact of sugarcane bagasse-derived biochar on heavy metal availability and

531

microbial activity: a field study. Chemosphere, 200, 274-282.

532

Ren, L., Cai, C., Zhang, J., Yang, Y., Wu, G., Luo, L., Huang, H., Zhou, Y., Qin, P., Yu,

533

M., 2018. Key environmental factors to variation of ammonia-oxidizing archaea

534

community and potential ammonia oxidation rate during agricultural waste composting.

535

Bioresour. Technol. 270, 278-285.

536

Song, D., Xi, X., Zheng, Q., Liang, G., Zhou, W., Wang, X., 2019. Soil nutrient and

537

microbial activity responses to two years after maize straw biochar application in a

538

calcareous soil. Ecotoxicol. Environ. Saf. 180, 348-356.

22

539

Sun, D.Q., Jun, M., Zhang, W.M., Guan, X.C., Huang, Y.W., Lan, Y., Gao, J.P., Chen,

540

W.F., 2012. Implication of temporal dynamics of microbial abundance and nutrients

541

to soil fertility under biochar application-field experiments conducted in a brown soil

542

cultivated with soybean, north China. Adv. Mater. Res. 384-394.

543 544

Tabatabai, M.A., Bremner, J.M., 1970. Arylsulfatase activity of soils 1. Soil Sci. Soc. Am. J. 34, 225-229.

545

Tang, J., Zhang, J., Ren, L., Zhou, Y., Gao, J., Luo, L., Yang, Y., Peng, Q., Huang, H.,

546

Chen, A., 2019. Diagnosis of soil contamination using microbiological indices: A

547

review on heavy metal pollution. J. Environ. Manage. 242, 121-130.

548

Touceda-González, M., Prieto-Fernández, Á., Renella, G., Giagnoni, L., Sessitsch, A.,

549

Brader, G., Galazka, R., 2017. Microbial community structure and activity in trace

550

element-contaminated soils phytomanaged by Gentle Remediation Options (GRO).

551

Environ. Pollut. 231, 237-251.

552

Wang, L., Chen, L., Tsang, D.C., Kua, H.W., Yang, J., Ok, Y.S., Ding, S., Hou, D., Poon,

553

C.S., 2019. The roles of biochar as green admixture for sediment-based construction

554

products. Cement. Concrete Comp. 103348.

555

Xian, Y., Wang, M., Chen, W.J.C., 2015. Quantitative assessment on soil enzyme activities

556

of heavy metal contaminated soils with various soil properties. Chemosphere, 139,

557

604-608.

558

Xu, Z., Yu, G., Zhang, X., Ge, J., He, N., Wang, Q., Wang, D., 2015. The variations in soil

559

microbial communities, enzyme activities and their relationships with soil organic

560

matter decomposition along the northern slope of Changbai Mountain. Appl. Soil Ecol.

561

86, 19-29.

23

562

Yang, J., Yang, F., Yang, Y., Xing, G., Deng, C., Shen, Y., Luo, L., Li, B., Yuan, H., 2016a.

563

A proposal of “core enzyme” bioindicator in long-term Pb-Zn ore pollution areas based

564

on topsoil property analysis. Environ. Pollut. 213, 760-769.

565

Yang, X., Liu, J., McGrouther, K., Huang, H., Lu, K., Guo, X., He, L., Lin, X., Che, L., Ye,

566

Z., 2016b. Effect of biochar on the extractability of heavy metals (Cd, Cu, Pb, and Zn)

567

and enzyme activity in soil. Environ. Sci. Pollut. Res. 23, 974-984.

568

Yoo, J.C., Beiyuan, J., Wang, L., Tsang, D.C., Baek, K., Bolan, N.S., Ok, Y.S., Li, X.D.,

569

2018. A combination of ferric nitrate/EDDS-enhanced washing and sludge-derived

570

biochar stabilization of metal-contaminated soils. Sci. Total Environ. 616, 572-582.

571

Zeng, G., Wu, H., Liang, J., Guo, S., Huang, L., Xu, P., Liu, Y., Yuan, Y., He, X., He, Y.,

572

2015. Efficiency of biochar and compost (or composting) combined amendments for

573

reducing Cd, Cu, Zn and Pb bioavailability, mobility and ecological risk in wetland

574

soil. Rsc. Adv. 5, 34541-34548.

575

Zeng, G., Zhang, J., Chen, Y., Yu, Z., Yu, M., Li, H., Liu, Z., Chen, M., Lu, L., Hu, C.,

576

2011. Relative contributions of archaea and bacteria to microbial ammonia oxidation

577

differ under different conditions during agricultural waste composting. Bioresour.

578

Technol. 102, 9026-9032.

579

Zeng, X., Xiao, Z., Zhang, G., Wang, A., Li, Z., Liu, Y., Wang, H., Zeng, Q., Liang, Y.,

580

Zou, D., 2018. Speciation and bioavailability of heavy metals in pyrolytic biochar of

581

swine and goat manures. J. Anal. Appl. Pyrol. 132, 82-93.

582

Zhang, J., Zeng, G., Chen, Y., Yu, M., Yu, Z., Li, H., Yu, Y., Huang, H., 2011. Effects of

583

physico-chemical parameters on the bacterial and fungal communities during

584

agricultural waste composting. Bioresour. Technol. 102, 2950-2956.

24

585

Figure caption

586

Fig. 1. Effect of amendments on the soil properties: (a) pH, (b) EC, (c) OM, (d) TOC, (e)

587

NH4+-N, (f) NO3–-N, (g) AP, (h) AK. Different letters above bars indicate significant

588

differences between mean values at each sampling occasion (P < 0.05).

589

Fig. 2. Effect of amendments on the concentration of CaCl2-extractable heavy metals: (a)

590

Cd, (b) Zn, (c) As, (d) Cu in soil. Different letters above bars indicate significant differences

591

between mean values at each sampling occasion (P < 0.05).

592

Fig. 3. Effect of amendments on the activities of soil enzyme: (a) dehydrogenase, (b)

593

catalase, (c) β-glucosidase, (d) invertase, (e) urease, (f) protease, (g) acid and (h) alkaline

594

phosphatase and (i) arylsulfatase. Different letters above bars indicate significant

595

differences between mean values at each sampling occasion (P < 0.05).

596

25

597 598

Fig. 1

26

Fig. 2

27

599

28

600

29

601 602

Fig. 3

603

30

604 605

Table 1 Physicochemical properties of experimental soil and amendments. Properties

606

Soil

Biochar

EC (ds.m-1) 0.21 ± 0.01 0.16 ± 0.01 pH (H2O) 5.98 ± 0.01 9.10 ± 0.02 OM (g kg-1) 62.33 ± 4.47 816.20 ± 4.23 -1 TOC (g kg ) 36.16 ± 2.59 473.44 ± 2.45 NH4+-N (mg kg-1) 38.32 ± 5.29 42.20 ± 1.39 -1 NO3 -N (mg kg ) 48.06 ± 4.24 6.98 ± 1.59 AP (mg kg-1) 33.45 ± 0.22 41.33 ± 0.71 TK (g kg-1) 5.67 ± 0.10 21.58 ± 1.35 -1 Total As (mg kg ) 55.07 ± 0.85 4.25 ± 0.16 Total Cd (mg kg-1) 0.48 ± 0.05 0.15 ± 0.01 -1 Total Cu (mg kg ) 50.15 ± 1.33 301.62 ± 0.89 Total Zn (mg kg-1) 100.55 ± 0.58 483.47 ± 13.75 3 Total pore volume (cm g ─ 0.05 ± 0.002 1) Specific surface area 0.69 ± 0.05 60.18 ± 3.12 Ash content (%) ─ 49.52 ± 1.23% Moisture (%) 17.95% 7.12% Numbers are presented as means ± standard deviations (SD)

31

Compost 7.99 ± 0.01 8.83 ± 0.02 274.41 ± 3.26 159.17 ± 1.89 354.56 ± 9.71 82.53 ± 4.27 95.64 ± 0.01 42.99 ± 2.37 6.04 ± 0.79 1.88 ± 0.09 29.93 ± 3.16 150.31 ± 11.45 ─ ─ ─ 23.13%

Table 2 Methods of soil enzyme activity assays. Enzyme

Substrate 2,3,5-triphenyl tetrazolium chloride (TTC)

Metabolite

Unit

References

Triphenyl formazan (TPF)

mg TPF g-1 soil h-1

Arif et al. (2018)

Catalase

H2O2

H2O

μmol H2O2 g-1 soil 24h-1

Johnson and Temple (1964)

β-glucosidase

p-nitrophenyl-β-D-glucopyranoside

p-nitrophenol (PNP)

μmol PNP g-1 soil 24h-1

liang et al. (2014)

Invertase

3,5-Dinitrosalicylic acid

Glucose

mg Glucose g-1 soil 24h-1

Urease

Urea

NH3-N

Protease

Na-caseinate

Tyrosine

mg NH3-N g-1 soil 24h-1 mg Tyrosine g-1 soil 24h-

Acid phosphatase

p-nitrophenol phosphate

Alkaline phosphatase Arylsulfatase

Dehydrogenase

Frankeberger and Johanson (1983) Yang et al. (2016a)

1

Touceda-González et al. (2017)

p-nitrophenol (PNP)

μmol PNP g-1 soil 24h-1

Arif et al. (2018)

p-nitrophenyl phosphate

p-nitrophenol (PNP)

μmol PNP g-1 soil 24h-1

Arif et al. (2018)

Potassium 4-nitrophenyl sulphate

p-nitrophenol (PNP)

μmol PNP g-1 soil 24h-1

Tabatabai and Bremner (1970)

32

Table 3 Correlations between soil physicochemical properties and CaCl2-extractable heavy metals. Cu

Zn

pH

pH

0.265

-0.217

0.076

-0.136

1

EC

0.904**

-0.607*

0.845**

-0.627*

0.155

1

OM

0.570

0.784**

0.133

-0.693*

0.366

0.285

1

NH4+-N

-0.008

-0.198

-0.006

-0.293

-0.663*

-0.010

0.144

1

NO3--N

0.269

0.039

0.136

0.004

0.614*

0.410

0.113

0.725**

1

AP

0.921**

-0.787*

0.713**

0.276

0.906**

0.516

0.047

0.387

1

AK

0.963**

-0.708*

0.880**

0.291

0.953**

0.420

-0.068

0.366

0.913**

Significant correlation: *P < 0.05; **P < 0.01.

33

OM

NO3--N

Cd

0.753** 0.706**

EC

NH4+-N

As

AP

AK

1

Table 4 Soil enzyme activities correlation matrix. As

Cd

Cu

Zn

pH

EC

OM

NH4+-N NO3--N

AP

AK

Dehydrogenase

0.929**

-0.661*

0.806**

-0.659*

0.186

0.928**

0.407

0.125

0.196

0.888**

0.931**

Catalase

0.935**

-0.646*

0.876**

-0.640*

0.078

0.931**

0.353

0.167

0.136

0.879**

0.917**

β-glucosidase

0.597*

-0.269

0.887**

-0.332

0.002

0.783**

-0.241

0.071

0.204

0.574

0.734**

Invertase

0.238

-0.209

0.279

-0.244

0.355

0.254

-0.011

-0.220

0.355

0.264

0.410

Urease

0.869**

-0.611*

0.848**

-0.612*

-0.035

0.874**

0.260

0.320

-0.006

0.838**

0.848**

Protease

0.237

-0.209

0.289

-0.237

0.357

0.223

-0.002

-0.207

0.286

0.226

0.398

Acid phosphatase

0.564

-0.261

0.871**

-0.340

-0.112

0.742**

-0.253

0.119

0.144

0.504

0.687*

Alkaline phosphatase

0.581*

-0.293

0.884**

-0.370

-0.126

0.707*

-0.229

0.169

0.054

0.488

0.696*

Arylsulfatase

0.729**

-0.410

0.906**

-0.455

-0.047

0.886**

-0.076

0.192

0.176

0.732**

0.825**

Significant correlation: *P < 0.05; **P < 0.01.

34

Abbreviation EC

Electrical conductivity

OM

Organic matter

TOC

Organic carbon

NH4+-N

Ammonium nitrogen

NO3–-N

Nitrate nitrogen

AP

Available phosphorus

AK

Available potassium

TK

Total potassium

607 608 609

35

610 611

612 613 614

Graphical Abstract

36

615 616

Highlights

617



Biochar and compost changed most soil physicochemical properties.

618



Combined application significantly reduced Cd and Zn availability.

619



Enzyme activities were activated by compost and inhibited by biochar.

620



EC, AK were important factors affecting metal availability and enzyme activities.

621



Availability of As, Cu, Cd and Zn affected dehydrogenase, catalase, urease.

622

37

623 624

Conflict of interest statement

625 626

The authors declare that they do not have any commercial or associative interest that

627

represents a conflict of interest in connection with the work submitted.

628

38