Calcined Eggshell Waste for Mitigating Soil Antibiotic-Resistant

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Calcined eggshell waste for mitigating soil antibiotic resistant bacteria/gene dissemination and accumulation in bell pepper Mao Ye, Mingming Sun, Yanfang Feng, Xu Li, Arthur Paul Schwab, Jinzhong Wan, Manqiang Liu, Da Tian, Kuan Liu, Jun Wu, and Xin Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00866 • Publication Date (Web): 22 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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Journal of Agricultural and Food Chemistry

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Calcined eggshell waste for mitigating soil antibiotic resistant bacteria/gene

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dissemination and accumulation in bell pepper

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Mao Ye,

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Wan, ╦ Manqiang Liu, § Da Tian, § Kuan Liu, § Jun Wu,§ and Xin Jiang *, †

*, †

Mingming Sun, §, || Yanfang Feng, ⱷ Xu Li, ‡ Arthur P. Schwab, || Jinzhong

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†

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Chinese Academy of Sciences, Nanjing 210008, PR China

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§

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Agricultural University, Nanjing 210095, PR China

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science,

Soil Ecology Lab, College of Resources and Environmental Sciences, Nanjing

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||

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88743, USA

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ⱷ

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Agricultural Sciences, Nanjing 210014, China

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‡

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Street, Lincoln, Nebraska 68588-6105, United States

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╦

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China, Nanjing 210042, People’s Republic of China

Department of Soil and Crop Sciences, Texas A&M University, College Station, TX

Institute of Agricultural Resources and Environment, Jiangsu Academy of

Department of Civil Engineering, University of Nebraska-Lincoln, 844 North 16th

Nanjing Institute of Environmental Science, Ministry of Environmental Protection of

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*Corresponding authors:

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Dr. Ye Mao and Prof. Dr. Jiang Xin

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E-mail address: [email protected], [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: The combined accumulation of antibiotics, heavy metals, antibiotic

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resistant bacteria (ARB)/genes (ARGs) in vegetables has become a new threat to

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human health. This is the first study to investigate the feasibility of calcined eggshells

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modified by aluminum sulphate as novel agricultural wastes to impede mixed

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contaminants from transferring to bell pepper (Capsicum annuum L.). In this work,

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calcined eggshell amendment mitigated mixed pollutant accumulation in bell pepper

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significantly; enhanced the dissipation of soil tetracycline, sulfadiazine, roxithromycin,

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and chloramphenicol; decreased the water-soluble fractions of antibiotics; and

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declined the diversity of ARB/ARGs inside the vegetable. Moreover, quantitative

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PCR analysis detected that ARGs levels in the bell pepper fruits significantly

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decreased to 10-10 copies/16S copies, indicating limited risk of ARG transferring

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along the food chain. Furthermore, the restoration of soil microbial biological

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function suggests that calcined eggshell is an environmentally-friendly amendment to

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control the dissemination of soil ARB/ARGs in the soil-vegetable system.

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KEYWORDS: antibiotic resistance genes; antibiotic resistant bacteria; calcined

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eggshells; impedance effect; bell pepper cultivation

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INTRODUCTION

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Human beings have a long, world-wide history of applying manure to enhance the

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cultivation of vegetables and crop cultivation

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organic production in recent decades, manure application has become even more

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important 3. Antibiotics are widely used in the livestock industry

1, 2

. With the growing emphasis on

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to treat diseases

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and promote growth 4. Most of the antibiotics are not fully metabolized by animals

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and are commonly be excreted with manure 2, 5. When manure is applied to the soil as

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fertilizer, antibiotics and their secondary metabolites could exert significant selective

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pressure on indigenous microorganisms, leading to an abundance of antibiotic

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resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in soil

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genetic elements (plasmids, integrons, and transposons) promote horizontal gene

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transfer between ARB and human pathogenic bacteria, posing a potential threat to

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public health

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tissue pores and epidermal wounds\ and colonize as antibiotic resistant endophytic

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bacteria (AREB) 2. Considering the symbiotic relationship between AREB and the

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plants throughout the whole growing phase, AREB thus brought along more directly

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threat against human health through food chain accumulation 9, 10. As a consequence,

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it is urgent to develop impedance practices to diminish the risk of ARG dissemination

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along the soil microbes/vegetables/human food chain, as well as decrease the

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accumulation of ARG/AREB inside vegetables.

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3, 6

. Mobile

7, 8

. Some indigenous ARB in soil could invade plants through surface

A promising approach to impeding transfer of ARB from soil to plants is the use 11, 12

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of environmentally friendly materials as a soil amendment

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biological waste with a porous surface, high absorption capacity, readily accessible,

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low price, and has been used previously in remediation studies 13-15. Physico-chemical

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modification of egg shells through calcining in the presence of aluminum sulfate

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resulted in the great improvement in the pore uniformity, structure regularity and

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diversity of surface functional groups

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. Eggshell is a

. Application of eggshell has been used

3



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successfully remove organic dyes and heavy metals from both water bodies and soils

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17-19

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transferring of antibiotics, ARB, and ARGs from soil to vegetables has not been

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investigated.

. However, the impeding effect of modified eggshells on the accumulation and

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In this study, eggshell modified by aluminum sulphate and calcining high

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temperature was used as an impeding material and was applied to an agricultural soil

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contaminated with antibiotics, ARB and ARGs. Bell pepper (Capsicum annuum L.)

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was selected as a representative of commonly consumed vegetables. The objectives of

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this research were to quantify the impact of calcined egg shell amendment as a soil

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amendment: i) on the concentrations of antibiotics and the populations of ARBs in

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soil; and, ii) on the transfer of contaminants and/or ARBs/ARGs from soil to bell

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pepper. To our best knowledge, this is the first study of its kind.

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MATERIALS AND METHODS

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Soil sampling and calcined eggshell preparation. Soil samples were collected in

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June, 2015 at Hengliang dairy farm in the suburban area of Nanjing, Eastern China

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(32°30’45’ N, 118°94’7’ E). According to the preliminary site investigation, the dairy

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farm has more than 10 thousand cows, leading to the production of approximate 50

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tons of fresh cow dung per day. Although most of the cow dung produced has been

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transported to the fertilizer plant to produce organic fertilizer through fermentation,

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there are still about 10 tons of fresh cow dung being directly dumped to the nearby

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farmland soils within a radius of 5 kilometers of the farm every day. Therefore, there 4


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is a potential risk of coexistence of high concentration of antibiotics and heavy metals

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in the surrounding agricultural soil for over 10 years (2005-2015).

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Typical farmland soil samples (0–15 cm, about 120 kg in total, each 10 kg) were

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collected from 12 locations around the farm. All soil samples were homogenized and

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grounded through a 2.0-mm sieve. The samples composed of 18.3% of sand, 64.3% of

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silt, and 17.4% of clay. The soil had a pH of 6.4, 25 mg organic matter/kg soil, 1.2 g

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kg-1 total N, 40.7 mg kg-1 hydrolyzable N, 0.4 g kg-1 total P and 29.1 mg kg-1 Olsen P.

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The predominant soil pollutants were cadmium (Cd), tetracycline (TC), sulfadiazine

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(SD), roxithromycin (RTM), and chloramphenicol (CAP), which were (9.9±0.7) ×10-1,

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2.3 ± 0.1, 3.4 ± 0.2, 1.4 ± 0.2, and 1.1 ± 0.1 mg kg-1, respectively.

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Waste eggshells were collected from a local cake workshop. Eggshells were

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immediately washed thoroughly with deionized water and dried at 105 °C for 24 h.

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All eggshells were ground and passed through a 2.0-mm sieve. Then 0.24 kg

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eggshells were added into aluminum sulphate solution prepared by dissolving 1.85 kg

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of Al2SO4.3H2O in 10 L deionized water 16. The mixture was adjusted to pH 3.0 using

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H2SO4 and continuously stirred for 12 h on shaker. After shaking, the mixture was

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transferred to Petri dish and dried at 105 °C for 12 h. Then dried mass was calcined at

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500 °C for 12 h in muffle furnace. The scanning electron microscopy

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photomicrographs of calcined eggshells are shown in Fig. S1. The textural properties

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and elemental contents for calcined eggshells were as follows: pH 7.8 (solid/deionized

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water = w/v = 1:5), 196.4 m2 g-1 specific surface area, 1.5×10-2 cm3 g-1 mesopore

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volume, 24.8 nm mesopore diameter, 32.6 wt % Ca2+, 0.8 wt % Mg2+, 1.6 wt % Fe3+, 5



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0.3 wt % Mn2+, 42.3 wt % Al3+, 2.2 wt % C, 0.2 wt % N, 0.1 wt % P, 0.1 wt % K and

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5.2 wt % S.

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Bell pepper cultivation. Soil microcosms were set up using a series of polyvinyl

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chloride cylindrical pots (bottom radius, 10 cm; height, 30 cm), each containing 7000

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g of air-dried soil. According to our preliminary experiment, the calcined eggshell

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amendment percentage at 0.5% (w/w) was the optimal one considering both

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impedance effect and the cost. Therefore, four treatments were set up as follows: (1)

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CK: control soil (initial contaminated soil), (2) SE: soil + 0.5% (w/w) calcined

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eggshell amendment, mixed thoroughly, (3) SB: soil + bell pepper cultivation, and (4)

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SEB: soil + 0.5% (w/w) calcined eggshell amendment + bell pepper cultivation.

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Triplicate samples were prepared.

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Bell pepper seeds were germinated and grown in moist perlite for 7 d. Then one

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uniform-sized seedling was transplanted to each designated pot. The pots were

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incubated in a greenhouse for 100 days at an average daily temperature of 22 ± 2 °C.

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The initial soil moisture content was adjusted to 65% of maximum field capacity.

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Every 3 days, deionized water was added according to the weight change of the

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microcosms. Every 30 days, approximately 15 g of soil was collected by taking five

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random soil samples up to a depth of 15 cm from each pot. After 150 days of

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cultivation, all soil and plant samples were placed in a small plastic bag and stored at

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4 °C for subsequent analysis. The weight of bell pepper tissues, total root surface area,

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and root activity, capsaicin, L-ascorbic acid, free amino acid, soluble sugar and 6


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protein in fruit were determined 20-22.

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Antibiotic and heavy metal analysis. The extraction procedures for soil TC, SD,

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RTM and CAP followed the methods described by Sun et al. 8. To investigate the

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changes in the different fractions of soil antibiotics (water-soluble, exchangeable,

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loosely bound, and tightly bound fractions, determined by the difference in binding

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formation between soil particles and antibiotics)

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placed in a 50-mL centrifuge tube and extracted with 40 mL of extract buffer (H2O-,

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CaCl2-, and Mcllvaine-). All the tubes were sonicated for 20 min and centrifuged at

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5000 rpm at 4 °C for 4 min. The supernatants were combined and filtered (pore size

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0.45 µm, PVDF membrane filter). The liquid was then purified and concentrated

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using a 60-mg Oasis HLB column from Waters (Milford, MA). Final extracts were

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analyzed using HPLC-MS-MS (Waters Acquity UPLC System) with electron spray

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ionization in a multiple reaction monitoring mode 25.

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23, 24

, one gram of soil sample was

Antibiotic contents in bell pepper tissues were measured following the methods 26

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described by Zhang et al.

. Specifically, the whole bell pepper tissues were

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thoroughly washed and divided into root, stem, leaf, fruit and seed. Each part of the

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tissues (0.5 g) were freeze-dried and ground to pass a 80 mesh sieve (0.177 mm). A

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mixed solution (10 mL) containing acetonitrile, phosphate buffer, and the bell pepper

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tissues were added into each centrifuge tube. The tubes were shaken in a shaker at 250

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rpm for 20 min, sonicated for 10 min, and then centrifuged at 7000×g for 10 min.

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Tetracycline was isolated from the samples using Oasis HLB extraction cartridges (6 7



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mL, 200 mg). The cartridge was rinsed with 6 mL 0.1% formic acid solution

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containing 5 mmol L-1 ammonium acetate, and retained antibiotics were eluted with

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6.0 mL methanol. This methanol fraction was re-dissolved in 10% methanol solution

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to a final volume of 1.0 mL prior to HPLC-MS-MS analysis. The average recoveries

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for TC, SD, RTM and CAP were between 93.1±1.2% and 104±3.2%.

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The total concentration of Cd was determined by digesting 2 g soil or plant

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samples using 50 mL HCl-HNO3-HClO4 (4:2:1, v/v/v), and was quantified with a

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Thermo flame Atomic Absorption Spectrophotometer

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method developed by the European Communities Bureau of Reference was performed

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on test soil samples to determine different fractions of soil Cd (exchangable,

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carbonate, organic, Fe-Mn oxide, and residual fraction) 28. The average recovery for

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Cd was 96.2±2.4%.

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. A sequential extraction

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Identification of ARB in bell pepper. After being separated from the twigs, bell

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pepper roots, stems, leaves, fruits and seeds were washed thoroughly with sterilized

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physiological solution (8.5 g L-1 NaCl) to eliminate adhering particles and surface

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microbes. The surface decontamination of bell pepper tissues was conducted

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following the description of Rahube et al.

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homogenized and incubated on R2A agar medium (0.5 g L-1 proteose peptone, 0.5 g

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L-1 casamino acids, 0.5 g L-1 yeast extract, 0.5 g L-1 dextrose, 0.5 g L-1 soluble starch,

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0.3 g L-1 dipotassium phosphate, 0.05 g L-1 Magnesium sulfate . 7H2O, 0.3 g L-1

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sodium pyruvate, 15.0 g L-1 Agar, pH 7.4) at 28 °C in the dark for 5 days. Visible

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.

The tissue pieces (1 cm²) were

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colonies of well-grown cultivable bacterial endophytes on the R2A medium were

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randomly selected for the analysis of antibiotic resistance (with TC, SD, RTM and

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CAP individual concentration of 10 mg L-1)

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universal sample tubes containing R2A medium (10 mL) at 28 °C and 160 rpm for 18

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hours, the cells were harvested and resuspended in 50 µL sterile distilled water. After

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being boiled at 100 °C for 10 min, and centrifuged at 12,000 rpm for 5 min

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micro liter lysate was used for polymerase chain reaction (PCR) to amplify 16S rDNA.

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When the amplified 16S rDNA fragment from each isolate was finalized, the obtained

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sequence was blasted against the collection of the nonredundant nucleotide sequence

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database of NCBI 6, 30.

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. After being incubated separately in

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, five

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Soil and bell pepper ARG quantification. A fast DNA SPIN Kit for Soil (MP

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Biomedicals, CA) was used to extract total DNA from 1.0 g of each soil sample. To

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determine the concentration and quality of the extracted DNA, spectrophotometric

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analysis (NanoDrop ND-2000c, Therome Fisher Scientific, Waltham, MA) and 1.5%

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agarose gel electrophoresis were used.

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After surface decontamination of the bell pepper tissues (described above),

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g fresh bell pepper tissues (root, stem, leaf, fruit and seed) were separately placed into

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a Stomacher Filtra-Bag (pore size of 330 µm) with 100 mL of sterile sodium

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metaphosphate buffer (2.0 g L-1, pH 7.0) and macerated. The macerate was then

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centrifuged at 12,000 rpm for 5 min. Then the pellet was used for DNA extraction

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according to the same procedure as that used for soil 29. Ten ARGs (tetW, tetM, tetB, 9


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tetQ, sulI, sulII, ermA, ermB, catI and catII) and the eubacterial 16S rRNA genes

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were detected and quantified by quantitative polymerase chain reaction (qPCR) using

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a SYBR Green approach 6. All qPCR reactions were repeated three times. The primer

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design can be found in Supporting Information Table S1.

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Microbial activity. Fast growing cultivable soil microorganisms were enumerated by

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the plating method 31. Physiological characteristics of the soil micro-community were

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measured following the Gram-negative microplate method 8.

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Data analysis. Data for eggshells as a soil amendment and the impact of bell pepper

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cultivation on antibiotic dissipation were analyzed by two-way ANOVA (SPSS 14.0

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software); means were compared by least significant difference (PSE>CK (Fig.1). In addition, two-way ANOVA

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indicated that an apparent synergistic interaction existed between calcined eggshell

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amendment and bell pepper cultivation (P

Calcined Eggshell Waste for Mitigating Soil Antibiotic-Resistant

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