XIANG Yang , XIANG Yan-ci , REN Hai , JIAN Shu-guang , WANG Rui-jiang , WANG Jie-ming , XIANG Jian-hua , HU Wei

1. Department of Landscape Architecture, School of Architecture and Art Design, Hunan University of Science and Technology, Xiangtan 411201, PRC;

2. Agricultural Bureau of Yongshun County, Yongshun 416700, PRC;

3. South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, PRC

Abstract A pot experiment was conducted to study the effects of biochar on physiological characteristics and accumulation of Cd and Pb of Lonicera japonica in Cd and Pb contaminated soil. The results showed that adding 2%~16% biochar could significantly increase the net photosynthetic rate, chlorophyll content and root activity of Lonicera japonica, and significantly reduce its SOD activity. When 6%~16% biochar was added, the contents of Cd and Pb in roots and stems of Lonicera japonica decreased significantly; when 2%~16% biochar was added, the contents of Cd and Pb in the leaves and flowers of Lonicera japonica decreased significantly. Adding more than 8% biochar can reduce the content of Cd and Pb in Lonicera japonica to a safe range level.

Key words Lonicera japonica; Biochar; Cd; Pb

1. Introduction

According to statistics, there are about 10 million hectares of arable land polluted by heavy metals such as Cd and Pb in China. Rice (

Oryza sativa

), maize (

Zea mays

) and honeysuckle (

Lonicera japonica

) growing in contaminated soil will absorb and accumulate heavy metals. Heavy metal

in-situ

immobilization remediation technology is a kind of heavy metal pollution control technology by adding modifiers to immobilize heavy metals in soil, reducing the bioavailability of heavy metals in soil, effectively preventing the migration and transfer of heavy metals into the food chain. The technology has become a research hotspot in the field of environmental science with short application cycle and economic benefits.

Biochar is a kind of highly aromatic refractory solid material produced by pyrolysis and carbonization of biomass under complete or partial hypoxia, which is a type of black carbon. Biochar has developed pores, large specific surface area, rich carbon and aromatic groups, and rich oxygen-containing functional groups on the surface. Biochar has the advantages of low price, strong adsorption, high stability and multi- functionality, and can absorb and retain pollutants. It can be used as an efficient adsorbent for the development and utilization of heavy metal contaminated land.

Previous studies have shown that biochar can affect the speciation and migration behavior of heavy metals in soil. However, there is no unified standard for reducing the degree of heavy metal pollution, application in production and dosage. In this paper, honeysuckle was used as a material to study the effects of different doses of biochar on physiological characteristics and Cd and Pb accumulation of

Lonicera japonica

in Cd and Pb contaminated soil by pot experiment, aiming to provide a basis for the safe use of heavy metal contaminated soil by biochar.

2. Materials and Methods

2.1. Test materials

The plant material was honeysuckle. The contents of Cd and Pb were 0.19 and 30.7 mg/kg, respectively. Pine biochar was purchased from the market, and the biochar with particle size of 0.5~1.0 cm was screened for reserve.

2.2. Test method

The soil was air dried naturally and it was grinded through 0.25 mm sieve for standby. 25 mg/kg Cd and 500 mg/kg Pb were added to the soil in the form of CdCl·2.5HO and Pb(NO), respectively. CdCl·2.5HO and Pb(NO)were dissolved in distilled water at the same time, and then added to soil. Deionized water was spraied, samples were mixed well, and then balanced for 50 d. Biochar was added to the contaminated soil at the ratios of 0, 2%, 4%, 6%, 8%, 10%, 12%, 14% and 16% (dry weight ratio of carbon to soil). The codes were B-0, B-2, B-4, B-6, B-8, B-10, B-12, B-14 and B-16, respectively, with B-0 as the control. The soil was put into a basin with a diameter of 15 cm and a depth of 20 cm, and each basin contained 4.0 kg of soil. The cuttings of

Lonicera japonica

were collected, and the branches with 15 cm length and uniform thickness were selected for indoor sand culture. When the vegetative leaves grew to 3~4 pieces, the seedlings with uniform growth were selected and transplanted into the basin, with 3 plants per basin. Six replicates per treatment. Watering was done regularly, honeysuckle plants were collected when flowering, plants were washed with tap water and distilled water, then were washed with deionized water, samples were dried, then dried at 105°C for 1 h, dried at 80°C for 8 h to constant weight.According to the method of ZHANG Z L

et al

., chlorophyll content, root activity and SOD activity were determined in 2017. Soil samples were digested with concentrated nitric acid + hydrofluoric acid + perchloric acid, and plant samples were digested with concentrated nitric acid + perchloric acid. The content of heavy metals was determined by atomic absorption spectrophotometry. The net photosynthetic rate (μmol/m·s) was measured by LI-6400 portable photosynthesis meter. The surface of biochar was wiped clean, and then cross cut with a blade, and the surface particles were blown off with an washing ear ball. The porous structure of biochar was observed under scanning electron microscope (ESEM) (Fig. 1). The area of large porosity (pore cross section 100~200 μm × 50~100 μm) was 39%, the area of small porosity (15~30 μm × 10~20 μm) was 44%, and the total area ratio of large and small pores was 1∶0.89.

3. Results and Analysis

3.1. Effects of biochar addition on net photosynthetic rate and chlorophyll content of Lonicera japonica

It can be seen from Fig. 2 that after adding biochar to the polluted soil, the net photosynthetic rate of

Lonicera japonica

leaves increased significantly, and the chloropyhⅡ content of leaves increased signi- ficantly, with significant differences (

P

<0.05); after adding 2%~16% biochar, the net photosynthetic rate of

Lonicera japonica

leaves increased by 27.6%~78.9% compared with the control; at the same time, the chlorophyll content of honeysuckle leaves increased by 31.8%~67.5% after adding 2%~16% biochar.

Fig. 1 Biochar and its structure

Fig. 2 Effects of biochar addition on net photosynthetic rate and chlorophyll content of Lonicera japonica

3.2. Effects of biochar addition on SOD activity and root activity of Lonicera japonica

It can be seen from Fig. 3 that the SOD activity of

Lonicera japonica

decreased by 15.9%~25.7% compared with the control after adding 6%~16% biochar, and the difference was significant (

P

<0.05); after adding 2%~16% biochar, the root activity of honeysuckle increased by 20.0%~37.8%, and the difference was significant (

P

< 0.05 ).

3.3. Effects of biochar addition on Cd and Pb acc- umulation in different parts of Lonicera japonica

3.3.1. Root

It can be seen from Fig. 4 that adding biochar to the polluted soil can effectively reduce the absorption and accumulation of Cd and Pb in the roots of

Lonicera japonica

. Compared with the control, adding 2% and 4% biochar could reduce the content of Cd in the root of

Lonicera japonica

, but the difference was not significant (

P

>0.05); the addition of 6%~16% biochar decreased the Cd content in roots by 27.6%~ 43.7%, and the difference was significant (

P

<0.05). Compared with the control, adding 2% biochar reduced the content of Pb in the root of

Lonicera japonica

, but the difference was not significant (

P

>0.05); however, adding 4%~16% biochar could reduce the content of Pb in the root of

Lonicera japonica

by 15.4%~48.9%, and the difference was significant (

P

<0.05).

3.3.2. Stem

It can be seen from Fig. 5 that adding biochar to the polluted soil can effectively reduce the accumulation of Cd and Pb in the stems of

Lonicera japonica

. Compared with the control, adding 2% and 4% biochar could not significantly reduce the Cd content in the stem of

Lonicera japonica

(

P

>0.05); the addition of 6%~16% biochar reduced the Cd content in the stem of

Lonicera japonica

by 40.7%~75.2%, and the difference was significant (

P

<0.05). Compared with the control, the addition of 2% biochar reduced the content of Pb in the stem of

Lonicera japonica

, but the decrease was not significant (

P

>0.05); the addition of 4%~16% biochar reduced the Pb content in the stem of

Lonicera japonica

by 29.0%~81.4%, with a significant decrease (

P

<0.05).

3.3.3. Leaf

It can be seen from Fig. 6 that the addition of biochar to the polluted soil can significantly reduce the contents of Cd and Pb in the leaves of

Lonicera japonica

. Compared with the control, adding 2%~16% biochar decreased the Cd content by 18.7%~52.4% and the Pb content by 13.6%~42.9% in the leaves of

Lonicera japonica

, and the decrease were significant (

P

<0.05).

3.3.4. Flowers

It can be seen from Fig. 7 that adding biochar to the polluted soil can significantly reduce the contents of Cd and Pb in flowers of

Lonicera japonica

. Compared with the control, adding 2%~16% biochar could reduce the content of Cd in flowers of

Lonicera japonica

by 56.1%~92.0%, with significant difference (

P

<0.05). After adding 8%~16% biochar, the content of Cd in flowers of

Lonicera japonica

was 0.20~0.37 mg/kg. Compared with the control, adding 2%~16% biochar could reduce the Pb content in flowers of

Lonicera japonica

by 22.2%~80.1%, with significant difference (

P

<0.05). After adding 6%~16% biochar, the content of Pb in folwers of

Lonicera japonica

was 1.55~ 4.04 mg/kg.

Fig. 3 Effects of biochar addition on SOD activity and root activity of Lonicera japonica

Fig. 4 Effects of biochar addition on Cd and Pb contents in roots of Lonicera japonica

Fig. 5 Effects of adding biochar on Cd and Pb content in stem of Lonicera japonica

Fig. 6 Effects of biochar addition on Cd and Pb contents in leaves of Lonicera japonica

Fig. 7 Effects of adding biochar on Cd and Pb contents in flowers of Lonicera japonica

4. Discussion and Conclusion

It has been reported that adding biochar can increase pH value of substrate, CEC and water holding capacity, and reduce the bioavailability of Cd, Pb and Zn. Cow dung biochar can effectively adsorb Pb in wa ter, and its adsorption efficiency is much higher than that of activated carbon. Pb in soil can be immobilized by biochar made from cow dung. Pb and P in biochar form insoluble Pb(PO)(OH) and are immobilized. The addition of biochar significantly increased soil pH, CEC and organic matter content, decreased soil Pb available content, significantly increased soybean yield, and reduced soybean absorption of As and Pb. It was found that biochar could effectively reduce the accumulation of Cd and Pb in roots, stems, leaves and flowers of

Lonicera japonica

. When 6%~16% biochar was added, the contents of Cd and Pb in roots and stems of

Lonicera japonica

decreased significantly (

P

<0.05). When 8%~16% biochar was added, the content of Cd in

Lonicera japonica

was 0.20~0.37 mg/kg; when 6%~16% biochar was added, the Pb content in honeysuckle was 1.55~4.04 mg/kg. According to the

Green Industry Standards for Import and Export of Medicinal Plants and Preparations

with Pb content≤5.0 mg/kg, Cd content≤0.38 mg/kg of limit, adding more than 8% biochar can reduce the Cd and Pb content in honeysuckle to a safe range level.When no biochar was added to the Cd and Pb contaminated soil, the contents of Cd and Pb in the root, stem, leaf and flower organs of

Lonicera japonica

were high. The two heavy metals caused biological toxicity to it, resulting in low root activity, low chlorophyll content and net photosynthetic rate, and high SOD activity. When biochar was added to the contaminated soil, the contents of Cd and Pb in roots, stems, leaves and flowers of

Lonicera japonica

decreased, which weakened the toxic effects of heavy metals on plants, increased root activity, chlorophyll content and net photosynthetic rate, and decreased SOD activity. Cd and Pb are highly toxic heavy metal elements. When plants are stressed by Cd and Pb, Cd and Pb will inhibit the activity of photosynthetic enzymes and the synthesis of chlorophyll, reduce the photosynthetic efficiency, and make the total amount of chlorophyll and photosynthesis show a downward trend. After Cd treatment, the total chlorophyll content and protein content of

Bacopa monnieri

decreased. When the content of heavy metals in the substrates is high, the plant cells will be damaged, the cell membrane lipid will be oxidized to produce MDA, the free radicals in the plant will increase, and the activity of peroxidase such as SOD will increase. Adding biochar in heavy metal contaminated soil can absorb and sequestrate heavy metals, reduce the bioavailability of heavy metals and reduce the toxicity of heavy metals to plants.Comprehensive consideration, adding biochar in Cd and Pb contaminated soil can effectively reduce the accumulation of Cd and Pb in root, stem, leaf and flower organs, significantly increase net photosynthetic rate, chlorophyll content and root activity, and significantly reduce SOD activity of

Lonicera japonica

; adding more than 8% biochar can reduce the content of Cd and Pb in flowers to a safe range level.