Microplastic particles increase arsenic toxicity to rice seedlings

Journal Pre-proof Microplastic particles increase arsenic toxicity to rice seedlings Youming Dong, Minling Gao, Zhengguo Song, Weiwen Qiu PII:

S0269-7491(19)32938-0

DOI:

https://doi.org/10.1016/j.envpol.2019.113892

Reference:

ENPO 113892

To appear in:

Environmental Pollution

Received Date: 4 June 2019 Revised Date:

23 December 2019

Accepted Date: 27 December 2019

Please cite this article as: Dong, Y., Gao, M., Song, Z., Qiu, W., Microplastic particles increase arsenic toxicity to rice seedlings, Environmental Pollution (2020), doi: https://doi.org/10.1016/ j.envpol.2019.113892. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical Abstract

Reduced Decrease biomass biomass Inhibit photosynthesis Inhibit root activity oxidative burst burst Destroy enzyme structure; Mechanical damage

hi In bi t

As (

As ( ); PS, PTEF

) uptake

1

Microplastic particles increase arsenic toxicity to rice seedlings

2

Youming Donga, Minling Gaob, Zhengguo Songb*, Weiwen Qiuc

3

a

4

300191, China

5

b

6

515063, China

7

c

8

Christchurch 8140, New Zealand

9

*Corresponding author: Tel: 0086 13920782195

10

Agro-Environmental Protection Institute, Ministry of Agriculture of China, Tianjin,

Department of Civil and Environmental Engineering, Shantou University, Shantou,

The New Zealand Institute for Plant and Food Research Limited, Private Bag 4704,

E-mail: [email protected]

11 12 13 14 15 16 17 18 19 20 21 22 1

23

Abstract: Hydroponic experiments were conducted to study the effects of

24

microplastic particles of polystyrene (PS) and polytetrafluoroethylene (PTFE) on

25

arsenic (As) content in leaves and roots of rice seedlings, and the changes in root

26

vigor and physiological and biochemical indicators under single or combined PS and

27

PTFE with As(III) treatment. Rice biomass decreased with increasing concentrations

28

of PS, PTFE, and As(III) in the growth medium. The highest root (leaf) biomass

29

decreases were 21.4% (10.2%), 25.4% (11.8%), and 26.2% (16.2%) with the addition

30

of 0.2 g L-1 PS, 0.2 g L-1 PTFE, and 4 mg L-1 As(III), respectively. Microplastic

31

particles and As(III) inhibited biomass accumulation by inhibiting root activity and

32

RuBisCO activity, respectively. The addition of As(III) and microplastic particles (PS

33

or PTFE) inhibited photosynthesis through non-stomatal and stomatal factors,

34

respectively; furthermore, net photosynthetic rate, chlorophyll fluorescence, and the

35

Chl a content of rice were reduced with the addition of As(III) and microplastic

36

particles (PS or PTFE). Microplastic particles and As(III) induced an oxidative burst

37

in rice tissues through mechanical damage and destruction of the tertiary structure of

38

antioxidant enzymes, respectively, thereby increasing O2- and H2O2 in roots and

39

leaves, inducing lipid peroxidation, and destroying cell membranes. When PS and

40

PTFE were added at 0.04 and 0.1 g L-1, respectively, the negative effects of As(III) on

41

rice were reduced. Treatment with 0.2 g L-1 PS or PTFE, combined with As(III), had a

42

higher impact on rice than the application of As(III) alone. PS and PTFE reduced

43

As(III) uptake, and absorbed As decreased with the increasing concentration of

44

microparticles. The underlying mechanisms for these effects may involve direct 2

45

adsorption of As, competition between As and microplastic particles for adsorption

46

sites on the root surface, and inhibition of root activity by microplastic particles.

47

Keywords: biomass, antioxidant enzymes, RuBisCO activity, physiological activity,

48

mechanisms

49

Capsule: 0.2 g L-1 PS or PTFE and As(III) coexisted treatment could aggravate As

50

toxicity to rice seedlings.

51 52 53

1. Introduction To date, 79% of the approximately 6.3 billion tons of plastic waste produced

54

worldwide have been discarded in landfills (Geyer et al., 2017). Once in the

55

environment, plastics are gradually decomposed to millimeter- and micrometer-sized

56

particles to form microplastics under the action of mechanical friction and biological

57

and photodegradation (Rochman et al., 2013).

58

The study of microplastics first focused on marine ecology and their toxic effects

59

on marine organisms, such as the discovery of microplastic particles in oysters

60

(Sussarellu et al., 2016). Rillig (2012) first focused on soil microplastic contamination

61

and found that the entry of microplastics into the soil affected soil properties, soil

62

function and biodiversity. The number of micro-plastic particles due to the application

63

of sludge, reportedly ranged from 1000 to 4000 per kilogram of soil in European

64

farmlands (Zubris and Richards, 2005). Similarly, microplastic content in soils of the

65

industrial zone in Sydney, Australia, is as high as 0.03% to 6.7% (Fuller and Gautam,

66

2016). The main source of micro-plastics in farmlands around the world is the 3

67

degradation of abandoned agricultural films extensively used for crop protection

68

against low ambient temperature and to minimize soil moisture loss under conditions

69

of high evaporative demand (Chen et al., 2013). Long-term coverage with plastic

70

films in Xinjiang agricultural topsoil (0-20 cm) reduced cotton production by as much

71

as 15% (Liu et al., 2014). Therefore, we speculated that microplastic particles have a

72

certain adverse effect on crop yield.

73

Arsenic (As) pollution has become a global environmental issue attracting

74

increasing attention. Indeed, the US Environmental Protection Agency has identified

75

As, as one of the five most harmful soil pollutants (Johnson and Derosa, 1995).

76

Humans mainly ingest environmental As through drinking water and food webs,

77

which can cause various diseases, such as skin damage, high blood pressure, nervous

78

problems, and even cancer (Martinez et al., 2011). As present in the environment

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mainly enters the human body through diet and drinking water.

80

An important staple food for people in northeastern and southern China, rice

81

shows a highly efficient As accumulating mechanism under flooding conditions, thus

82

allowing As to enter the plant tissues (Ma et al., 2017). Therefore, As pollution in rice

83

is a serious issue in China. Some surveys have shown that the content of As in cereal

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crops in China ranges from 70 to 830 µg kg-1. In some mining areas with serious As

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pollution levels in Chenzhou, As content in rice kernels can reach 500–7500 µg kg-1

86

(Liao et al., 2005). In fact, there have been studies on the absorption and the

87

toxicological mechanism of As, while others have focused on various methods for

88

reducing the toxicity of As in rice (Saifullah et al., 2018); however, to our knowledge, 4

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there are no studies on the effects of the currently emerging problem of pollutant

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micro-plastic particles in combination with traditional As pollution in rice. The most

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paddy fields in the south of China use river water as irrigation source in which

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microplatics pollution is progressing, and such microplastics-polluted irrigation water

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could interact with As to form combined contimatants in the As-contaminated soil.

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Plastic is a high molecular weight polymer, a long-chain molecule composed of

95

repeating structural monomers. Long-chain molecules show strong van der Waals

96

forces and, because of long-term weathering in the environment, micro-plastics

97

acquire special surface characteristics (i.e., high specific surface area, porosity, and

98

amorphous structure), thus becoming readily adsorbed pollutants (Brenneckea et al.,

99

2016). Roachman et al. (2013) found that the absorption of polycyclic aromatic

100

hydrocarbons (PAHs) by glassy polystyrene (PS) was higher than that by other

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microplastic particles. This material is highly adsorptive, mainly because the benzene

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ring increases the distance between adjacent polymer chains, making it easier for

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chemicals to diffuse into the polymer. Metal ions were found to interact with charged

104

or neutral regions on the surface of polyethylene (PE) microplastics (Ashton et al.,

105

2010). Although these studies have initiated an understanding of the toxicity of

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microplastics and class A, B and borderline metal (Duffus., 2002) complexes in

107

organisms, the effects of microplastic particles in combination with As on rice have

108

not been studied.

109 110

Polystyrene particles have a density similar to that of water, thus they can be suspended in an aqueous environment. Currently, polystyrene is already quite 5

111

abundant in the environment, and the global demand for polystyrene is expected to

112

reach the overall benchmark of 23.5 million tons in 2020 (Hansen et al., 2015). In turn,

113

polytetrafluoroethylene (PTFE) has a density of approximately 2.2, which is much

114

higher than that of water. It is chemically highly stable and has greater resistance to

115

high and low temperature, whereby, it has become the most consumed fluororesin

116

(Aderikha and Shapovalov, 2010). At present, As pollution in rice fields in southern

117

China is widespread and increasingly severe. Due to their slow degradation rate,

118

microplastics tend to accumulate and migrate in the soil, posing an additional threat to

119

biosafety. In recent years, microplastics have been a research hotspot in the field of

120

environmental science. The studies have been conducted to investigate their toxicity

121

on aquatic organisms and their interaction with other pollutants in water. However,

122

there are no reports on the effects of microplastic particles on As uptake by rice and

123

physiological and biochemical aspects of rice under As stress. Therefore, a

124

hydroponic experiment, easily controlled and plainly observed, is needed to study the

125

mechanism whereby microplastics impact As absorption by rice. The purpose of this

126

study was to conduct such experiments to study the effects of PS and PTFE particles

127

on As content in leaves and roots, as well as changes in root vigor and physiological

128

and biochemical indicators in rice seedlings, and to clarify the mechanism underlying

129

the effects of microplastic particles on As absorption by rice, along with its effects on

130

rice growth.

131

2. Materials and methods

132

2.1. Materials and reagents 6

133

Polystyrene (PS) and Polytetrafluoroethylene (PTFE) resin pellets were

134

purchased from the China National Petroleum Corporation Dushanzi Petrochemical

135

Company. A standard solution of As(III) (1000 mg·L-1) was supplied by

136

Sigma-Aldrich (St. Louis, USA). Hydroponic culture was carried out using seeds of

137

rice genotype T-705 (T705) (purchased from Hunan Longping Seed Co., Ltd.). The

138

reagent used to prepare the Hoagland nutrient solution was purchased from Shanghai

139

Aladdin Biochemical Technology Co., Ltd. Ultrapure water (resistivity = 18 MΩ·cm)

140

was obtained from a Milli-Q (Millipore) water purification system to use as the

141

growth medium.

142

2.2. Hydroponic experiment

143

2.2.1 Seed pretreatment

144

Selected fully developed rice seeds of uniform size were soaked in a 30% H2O2

145

solution for 15 min and then rinsed with distilled water. The seeds were placed on a

146

nursery tray in a constant temperature incubator for 48 h (37 °C) in the dark, and then

147

transferred to an artificial climate chamber (temperature: 25 °C, air humidity: 60%,

148

illumination time: 18 h·d-1) (Huang et al., 2017).

149

2.2.2 Preparation of Microplastic Particles and As(III) Suspension

150

PTFE and PS microplastic particles with different sizes were reprocessed by a

151

ball mill at Qinhuangdao Taiji Ring Nano Products Co., Ltd. The PTFE resin particles

152

were ball milled for 4 h using a nano-Ferris mill (Dong et al., 2019); a scanning

153

electron micrograph of the milled particles is shown in Supplementary Figure S1.

154

Different concentrations (0, 1.6, 3.2, or 4.0 mg·L-1) of As(III) were prepared in 7

155

1/10 Hoagland nutrient solution; 500 mL of each of these solutions were placed in

156

500-mL black PVC pots; concentrations of PS and PTFE with an average particle size

157

of 10

158

microplastic particles were added at 0.04, 0.1, or 0.2 g L-1 to 1.6, 3.2, and 4.0 mg L-1

159

As solution, respectively, and additionally As, PS and PTFE single treatment were set

160

up according to the above concentrations (Table S1). The black pots were placed in an

161

ultrasonic ice bath for 30 min. The solution pH was adjusted to 5.5-6.0 with

162

HCl/NaOH in order to inhibit the growth of pathogenic bacteria and to promote rice

163

growth.

164

2.2.3 Rice seedling exposure to experimental treatments

m were set at 0.04, 0.1, or 0.2 g L-1. Polystyrene and polytetrafluoroethylene

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Rice seedlings were grown in the nutrient solution for 10 days, rinsed with

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deionized water, and transferred to the above-mentioned PVC pots (18 seedlings per

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pot) containing the suspension of micro-plastic granules and As(III). To prevent

168

agglomeration of the microplastics, the PVC pots were sonicated every 12 h. Samples

169

were collected after 7 d of culture in an artificial climate chamber (Abdel-Haliem et

170

al., 2017).

171

2.3 Rice biomass accumulation

172

The roots were immersed in a 20 mmol·L-1 EDTA-2Na solution for 15 min to

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remove surface-adsorbed microplastic particles and As (III), and then rinsed. Next,

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seedlings were divided into roots and shoots (blades), and oven-dried for 0.5 h at

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105 °C, and then at 75 °C to constant weight.

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2.4 Photosynthesis parameters and pigments 8

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2.4.1 Photosynthesis and chlorophyll fluorescence parameters

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Net photosynthesis rate (Pn, µmol CO2 m-2 s-1), intercellular CO2 concentration

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(Ci, µmol CO2 mol-1), stomatal conductance (gs, µmol H2O m-2 s-1), and transpiration

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rate (Tr, mmol H2O m-2 s-1) were determined with a portable system (LI-6400XT;

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Li-Cor, Lincoln, Nebraska, USA) on the middle portion of the second leaf (from the

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top) of previously selected seedlings. Measurements were performed as previously

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reported (Gao et al., 2016).

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After placing the leaves in the dark for 30 min, the relevant parameters were

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determined using a chlorophyll fluorometer (PAM 2000; Heinz Walz GmbH,

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Effeltrich, Germany). Measurements were performed as previously reported (Gao et

187

al., 2016).

188

2.4.2 Photosynthetic pigments

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Chlorophylls from leaves were extracted with an acetone: absolute ethanol

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solution (2:1 v/v). The absorbance of sampled extracts was measured at 665 and 649

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nm using a UV-vis spectrophotometer (Shimadzu UV-1800, Japan). Specific methods

192

of operation were as previously reported (Gao et al., 2019).

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2.5 Antioxidant capacity analysis

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Rice tissues were thoroughly grounded under liquid nitrogen. Ground tissues and

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extracts were mixed in a ratio of 1:10 and homogenized in an ice bath. Homogenates

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were centrifuged at 10,000 × g for 4 min at 4 °C and then placed on ice for testing,

197

subsequent enzyme activity assays were conducted according to the method of Park et

198

al. (2011). 9

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The superoxide anion (O2•-) reacts with hydroxylamine hydrochloride to form

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NO2-. Under the action of p-aminobenzenesulfonic acid and α-naphthylamine, NO2-

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forms an azo compound, which has an absorption peak at 530 nm, according to which

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the content of O2•- in the sample can be calculated. Titanium sulfate and H2O2 form a

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precipitate of yellow titanium oxide complex with characteristic absorption peak at

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415 nm.

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Superoxide anions (O2•-) are produced by the reaction of purine and purine

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oxidase. The superoxide anion reduces nitro-blue tetrazolium to form blue formazan

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with a characteristic absorption peak at 560 nm; SOD can inhibit the formation of

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formazan. Therefore, the absorption intensity of formazan can be used to estimate

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SOD activity. Similarly, H2O2 shows a characteristic absorption peak at 240 nm; CAT

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decomposes H2O2, thus reducing absorbance at 240 nm, whereby CAT activity can be

211

estimated. Finally, tissue thiobarbituric acid reactant (TBARS) is condensed with

212

thiobarbituric acid to form a red product with maximum absorption at 532 nm. The

213

lipid content in the sample can be estimated after colorimetric determination. The

214

difference between the absorbance at 600 nm and the absorbance at 532 nm can be

215

used to calculate TBARS content.

216

2.6 Root system activity assay

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The determination and calculation of root system activity were carried out using

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the Kit available from Shanghai Yuanye Biotechnology Co., Ltd., according to

219

manufacturer instructions. 0.1 g of rice root sample was weighed out, washed, dried,

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and placed in a centrifuge tube. Then, 10 mL of 2,3,5-triphenyltetrazolium chloride 10

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(TTC) working solution was added and the mixed samples were incubated at 37 °C

222

for 1 h.; then, 2 mL of TTC stop solution was added to each sample. The roots were

223

drawn from the reaction vials, dried on filter paper, and thoroughly homogenized by

224

adding 3.5 ml of ethyl acetate to extract TTF. Root residues were washed several

225

times, and the washing solution and the red extract were transferred to a centrifuge

226

tube, before adding ethyl acetate to a final volume of 10 ml. The absorbance was

227

measured at 485 nm.

228

2.7 Ribulose bisphosphate carboxylase oxygenase (RuBisCO) activity

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Samples (0.1 g) were added with 1 mL of extract and ultrasonically crushed after

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homogenization in an ice bath. The conditions for sonication were as follows:

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temperature was 0 °C, power setting was 200 W, crushing period was 3 s, interval was

232

7 s, and total time was 1 min. Samples were centrifuged for 10 min at 8000 × g at

233

4 °C. The supernatant was used to assay RuBisCO activity by measuring the change

234

in absorbance at 340 nm.

235

2.8 As in rice

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Root and leaf samples (0.25 g dry weight) were placed in a Teflon digestion tube;

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next, 7 mL of nitric acid was added to each sample. The tubes were sealed and placed

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in the Teflon digestion tube for 8 h. Then, all the sample mixtures were placed in an

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electric heat digestion furnace (ED54, Lepertyco, USA) at 110 °C for 4 h, until all

240

samples were completely digested. Determination of As in samples was performed

241

using an atomic fluorescence spectrometer (AFS-9760, Beijing Pengjiang Haiguang

242

Instrument Co., Ltd.) (Huang et al., 2018). 11

243 244

2.9 Statistical analyses The experiment was laid in a completely randomized design with three

245

biological replicates. SPSS software (version 18.0, USA) was used to perform

246

one-way analysis of variance (ANOVA) of the data, followed by the Tukey´s test to

247

separate significantly different means. The Origin software (version 8.0, USA) was

248

used to prepare histograms.

249

3. Results

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3.1 Effects of microplastic and As(III) treatments on rice seedling biomass

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Microplastic particles (PS and PTFE) and As(III) applied as alternative single

252

pollution treatments inhibited biomass accumulation in roots and leaves of rice

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seedlings; further, a negative correlation between biomass and pollutant concentration

254

was observed (Table 1). When the concentration of PS or PTFE in the culture medium

255

was 0.2 g L-1 and the concentration of As(III) was 4 mg L-1, the maximum inhibition

256

of roots (leaves) biomass in rice seedlings was 21.4% (10.2%), 25.4% (11.8%), and

257

26.2% (16.2%), respectively. When As was combined with 0.04 and 0.1 g L-1 PS and

258

PTFE, respectively, the reduction in root and leaf biomass was lower than that of As

259

treatment alone, indicating that PS and PTFE countered the negative impact of As on

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biomass accumulation at these concentrations. Root and leaf biomass were reduced to

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a larger extent when As was combined with PS and PTFE at 0.2 g L-1 each, than when

262

applied alone. The effect of PTFE on rice biomass accumulation was slightly larger

263

than that of PS.

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3.2 Effects of microplastic and As(III) treatments on chlorophyll 12

265

None of the treatment groups in this experiment had a significant effect on

266

chlorophyll b content in rice (p > 0.05) (Table 1). PS, PTFE, and As(III) all showed

267

the same effect on rice; the higher the pollutant concentration, the more significant the

268

effect on Chl a and total Chl contents. Chl a (total chlorophyll) content was mostly

269

reduced at 0.2 g L-1 PS, 0.2 g L-1 PTFE, and 4 mg L-1 As(III); under such treatment,

270

Chl a (total chlorophyll) was reduced by 14.9% (13.2%), 19.3% (15.0%), and 20.7%

271

(16.1%), respectively. When the concentrations of PS and PTFE in the culture

272

medium were 0.04 and 0.1, respectively, they effectively inhibited the damage by

273

As(III) to chlorophyll, but when PS and PTFE concentrations were both 0.2 g L-1, in

274

combination with As(III), then the damage to Chl was higher than that caused by

275

As(III) treatment alone.

276

3.3 Effects of microplastic and As(III) treatments on photosynthesis and

277

chlorophyll fluorescence parameters

278

When rice seedlings were separately subjected to PS, PTFE, or As(III) stress, Net

279

photosynthesis rate (Pn), stomatal conductance (gs), maximum photochemical

280

efficiency (Fv/Fm), and electron transfer rate (ETR), as well as Tr were all increasingly

281

inhibited with increasing pollutant concentration. When the concentration of As was 4

282

mg L-1 and the concentration of PS and PTFE were 0.2 g L-1, pn; gs; Fv/Fm; ETR; Tr

283

decreased by 23.5%, 8.9% and 12.7%; 25.7%, 11.4% and 17.1%; 22.4%, 6.6% and

284

6.9%; 17.5%, 10.0% and 13.8%; 29.3%, 12.2% and 14.6% compared to control,

285

respectively (Table 2). PS and PTFE had little effect on rice intercellular CO2

286

concentration (Ci), which was significantly reduced only under the highest 13

287

concentration of PTFE in the hydroponic solution (p < 0.05), whereas it increased

288

with increasing As(III) concentration.; indeed, when the concentration of As(III) was

289

3.2 and 4.0 mg L-1, Ci increased by 15.3% and 28.3% respectively, compared to

290

controls. Addition of 0.04 and 0.1 g L-1 PS and PTFE particles to the As-contaminated

291

medium alleviated the effect of As(III) on rice photosynthesis; conversely, addition of

292

0.2 g L-1 PS and PTFE increased the inhibition of rice photosynthetic capacity.

293

3.4 Effects of microplastic and As(III) treatments on O2.- and H2O2 production

294

Exogenous addition of PS, PTFE, and As(III) increased the content of O2- and

295

H2O2 in rice roots and leaves, and showed a positive dose-effect relationship (Figure

296

1). At 4 mg L-1 As(III) and 0.2 g L-1 PS and PTFE, root (leaf) O2.- content in rice

297

increased by 85.3%, 27.1% and 31.9% (71.0%, 17.4% and 18.0%) compared to

298

controls, respectively. More significantly, root (leaf) H2O2 of rice increased by 55.8%

299

and 65.6%, 166.0% (23.4% and 25.8%, 114.7%) compared with controls. The effect

300

of As(III) on the production of O2.- and H2O2 in rice tissues was higher than that of PS

301

and PTFE. However, the effect of As(III) in combination with 0.04 and 0.1 g L-1

302

PTFE or PS on the production of O2- and H2O2 in rice was lower than that of As

303

treatment alone. At 0.04 and 0.1 g L-1 PTFE or PS, combined with As(III), O2.- and

304

H2O2 contents in rice tissues were lower than those observed under As treatment

305

alone.

306

3.5 Effects of microplastic and As(III) treatments on antioxidant activity

307

When exogenous PS and PTFE were less than 0.1 g L-1, SOD and CAT activities

308

in rice tissues of treated seedlings were slightly higher than those recorded for control 14

309

seedlings. Further, when PS and PTFE concentrations were 0.2 g L-1, SOD and CAT

310

activities decreased significantly. As(III) treatment alone caused a significant decrease

311

in SOD and CAT activities in rice tissues, and the higher the concentration, the more

312

significant the decrease in enzyme activities (Figure 2). Addition of 0.04 and 0.1 g L-1

313

PTFE and PS to the As-contaminated growth medium reduced the effect of As(III) on

314

SOD and CAT activities, whereas addition of 0.2 g L-1 PS and PTFE caused more

315

damage to SOD and CAT activities than As contamination alone.

316

3.6 Effects of s microplastic and As(III) treatments on TBARS

317

The TBARS accumulation trend in rice roots and leaves was affected by single

318

and combined As(III) treatment with PS or PTFE and was similar to that of O2- and

319

H2O2 (Figure 3). When PS and PTFE concentrations in the growth medium were 0.2 g

320

L-1 in combination with As(III) at 4 mg L-1, the root (leaf) TBARS increased by 24.5%

321

and 32.7%, 65.4% (21.1% and 25.9%, 62.1%), respectively.

322

3.7 Effects of microplastic and As(III) treatments on RuBisCO and root activity

323

PS and PTFE particles had a weaker effect on RuBisCO, but showed a severe

324

impact on root activity (Figure 4), whereas As(III) inhibited RuBisCO activity in a

325

dose-dependent manner. The As(III) 1.6 mg L-1 treatment had no significant effect on

326

root activity (p > 0.05), but root activity decreased with increasing As(III)

327

concentration. The trends of RuBisCO and root activity in leaves and roots, were

328

affected by separate or combined As(III) and PS or PTFE treatment, respectively,

329

which had a similar effect on SOD and CAT activities.

330

3.8 Effects of microplastics on As(III) uptake 15

331

The As(III) content in rice leaves and roots increased with increasing As(III)

332

concentration in the growth medium (Figure 5), but As was not detected in rice

333

seedlings of the control group, PS treatment group, and PTFE treatment group. PS and

334

PTFE effectively reduced As(III) content in rice tissues in a concentration-dependent

335

manner. There was no significantly different effect on As(III) content in roots and

336

leaves between PS and PTFE addition when As(III) was 1.6 mg L-1 in the solution.

337

However, after As(III) concentration reached 3.2 mg L-1 in the solution, PS addition

338

greatly reduced As(III) content in the roots than PTFE addition.

339

reduction in the roots, As(III) content in the leaves also significantly decreased by the

340

PS addition than PTFE addition in the 4.0 mg L-1 As(III) solution.

341

4 Discussion

342

Apart from As(III)

In this study, we found that the addition of PS and PTFE inhibited the absorption

343

of As(III) by rice; furthermore, addition of 0.04 and 0.1 g L-1 PS and PTFE reduced

344

the effects of As(III) on photosynthesis and antioxidant activity of rice; however, the

345

combination treatment of 0.2 g L-1 PS and PTFE, and 4.0 mg L-1 As(III) led to higher

346

effects on the photosynthesis and antioxidant activity of rice than those by As(III)

347

treatment alone. The effects of single or combined treatment with PS or PTFE and

348

As(III) on rice seedlings were reflected as an inhibition of biomass accumulation, but

349

the mechanism of inhibition may be different in each case. The mechanism may be (i)

350

accumulation in the epidermis or phloem of rice roots, whereby reduced root activity

351

affected nutrient uptake, and (ii) As(III) interfered with rice photosynthesis (Tripathi

352

et al., 2017). 16

353

The absorption of As by rice roots was significantly different due to changes in

354

root surface area, root activity, and transpiration. In the soil, As is adsorbed on the

355

root surface upon activation in the rhizosphere, and then transported into the plant

356

root through the apoplastic pathway and the symplastic pathway laterally (short

357

distance) (Redjala et al., 2010). Therefore, we speculate that PTFE and PS may be

358

adsorbed on to the surface of rice roots due to their hydrophobicity (Ziccardi et al.,

359

2016). During the experiment, PS and PTFE adsorbed on the surface of rice roots

360

were visible to the naked eye, thus they competed with As for the adsorption site of

361

As(III) or affected the absorption of As(III) by affecting root activity and

362

transpiration.

363

The addition of PS, PTFE, and As(III) reduced the photosynthetic rate compared

364

to the controls. In general, there are two types of factors responsible for the control of

365

the photosynthesis rate, namely, stomatal factors (i.e., stomatal conductance) and

366

non-stomatal factors (i.e., biochemical control of photosynthesis) (And and Sharkey,

367

2003). We hypothesized that PTFE and PS affected photosynthetic rate through

368

stomatal factors, while As(III) affected it through non-stomatal factors. The

369

transpiration rate change caused by PS, PTFE, and As(III) treatments altered the

370

response of the guard cells, leading to partial closure of stomata and a decrease in

371

stomatal conductance. The decrease in transpiration rate could be attributed to the

372

inhibition of root activity under PS, PTFE, and As(III) combined treatment, thus

373

reducing the ability to absorb water (Cseresnyés et al., 2014). As(III) may affect

374

photosynthesis through the following non-stomatal factors: (i) thylakoid membrane 17

375

damage, (ii) reduction of photosynthetic pigments (e.g., chlorophyll a, chlorophyll b

376

and total chlorophyll content in cells), and (iii) reduced activity of

377

photosynthesis-related enzymes (Tseng et al., 2018).

378

The decrease in chlorophyll content observed in this study was mainly caused by

379

the change in Chl a content. Therefore, after adding PS, PTFE, and As(III), the

380

content of Chl a decreased, and it can be inferred that the ratio of Chl a to Chl b

381

decreased as well. Exogenous additives may cause an "oxidative burst" in rice tissues,

382

thereby damaging chloroplasts and thylakoids, which in turn would result in a

383

decrease in chlorophyll content (Rossi et al., 2017). As can destroy chlorophyll

384

structure, hinder the synthesis of chlorophyll, and accelerate the decomposition of

385

chlorophyll (Várallyay et al., 2015). The findings of the present study indicated that

386

after exogenous addition of PS, PTFE, and As(III), LHC in rice leaves was damaged,

387

and the utilization of light energy and photosynthesis were reduced, thereby affecting

388

normal plant growth.

389

The decrease in Fv/Fm and ETR indicated that under PS, PTFE, and As(III) stress,

390

photoinhibition occurred in rice leaves due to damage to the PSII reaction center

391

(Ögren and Sjöström, 1990). When plants are subjected to xenobiotic stress, a large

392

amount of active oxygen may be generated in the tissue. Highly reactive oxygen

393

species (ROS) first attack the non-primary electron acceptor pheophytin (Pheo)

394

located on the D2 protein, and then the reaction center pigment molecule P680,

395

thereby making the PSII reaction center partially closed and thus, losing charge

396

separation (Fufezan et al., 2002). Inhibition of charge transfer leads to a decrease in 18

397

the number of Chl a molecules returned from the excited state to the ground state, and

398

consequently it reduces the content of ground state Chl a molecules and

399

photosynthetic efficiency (Powles, 2003). Piršelová et al. (2016) studied the toxic

400

effects of As (5 mg kg-1) in the soil on the growth and photosynthesis of two soybean

401

(Glycine max (L.) Merr.) varieties, namely, Bólyi 44 and Cordoba. Consistently with

402

our results, after 10 days of cultivation, maximum quantum yield of PSII (Fv/Fm) was

403

significantly lower.

404

He et al. (2004) found that when the leaves of mung bean seedlings were

405

exposed to UV-B radiation, the increase in leaf H2O2 content caused a decrease in

406

RuBisCO content. In the present study, the effect of As(III) on rice RuBisCO activity

407

was higher than that of PS or PTFE; therefore, As(III) likely affected biomass

408

accumulation in rice seedlings mainly through its effects on RuBisCO and the other

409

photosynthetic parameters, such as Pn, gs, Fv/Fm and ETR, and ultimately, on carbon

410

assimilation.

411

On the basis of our experimental results, we can speculate that under As(III)

412

stress, antioxidant enzymes SOD and CAT were damaged, whereby ROS could not be

413

timely removed, thus resulting in ROS accumulation and in membrane lipid

414

peroxidation, thereby resulting in excessive TBARS and cell membrane damage. The

415

reasons for the decrease in antioxidant enzyme activity caused by As include the

416

action of As on changes of tertiary structure of enzyme proteins (Ajees et al., 2012);

417

additionally, As inhibits the expression of antioxidant enzyme proteins, thus causing a

418

reduction in antioxidant enzyme half-life (Jobby et al., 2016). The mechanism by 19

419

which PS and PTFE trigger an oxidative burst in plant tissues is different from that of

420

As(III). These substances cannot directly act on cells, which may cause mechanical

421

damage of roots to produce ROS (Minibayeva et al., 2015), and then increase the

422

activity of ROS-induced antioxidant enzymes. ROS content and SOD and CAT

423

activities under PS and PTFE treatments at 0.04 and 0.1 g L-1, respectively, were

424

higher than in controls, indicating that rice responded to excess ROS by increasing

425

enzyme activity. The production of ROS under 0.2 g L-1 PS and PTFE may exceed

426

cell tolerance, consequently causing cell damage and inhibiting the expression of

427

antioxidant enzymes. The increase of TBARS indicated that membrane lipid

428

peroxidation occurred in rice tissues under PS, PTFE, and As(III) treatments, in a

429

concentration-dependent manner.

430

Both microplastic particles and As(III) caused an "oxidative burst" in rice tissues,

431

and the addition of low doses of PS and PTFE to As(III)-contaminated growth media

432

reduced root absorption of As(III), thereby reducing oxidative damage in rice.

433

Addition of high doses of PS and PTFE reduced As(III) content in rice tissues, but it

434

increased oxidative damage due to mechanical damage to rice roots.

435

Root activity is an overall indicator of the absorptive function of plants that so

436

strongly influences root growth, metabolism and absorption, and ultimately, growth

437

and development of aboveground parts. Under PS and PTFE treatment, root activity

438

of rice decreased significantly; this in turn decreased transpiration and As absorption.

439

On the other hand, As showed a significant effect on rice root activity only at high

440

concentration. When environmental factors are not conducive to root development, 20

441

root activity is hampered due to environmental stress-induced formation of peroxides

442

and oxygen free radicals in the roots or other plant organs that may threaten cell

443

membrane structure.

444

PS and PTFE affected As uptake in rice via three distinct pathways: direct

445

adsorption of As; competition with As for adsorption sites on the root surface; and

446

inhibition of root activity. As(III) entering plant tissues can induce ROS accumulation

447

by destroying the structure of antioxidant enzymes. As(III) and excess ROS impaired

448

rice chloroplasts, PSII reaction center and RuBisCO activity, thereby, negatively

449

affecting rice photosynthesis and biomass accumulation. PS and PTFE mainly caused

450

mechanical damage to the roots, reduced root vigor and transpiration, and caused a

451

large amount of ROS to be produced, which reduced the ability of plants to absorb

452

nutrients and water, thereby reducing photosynthetic capacity and biomass

453

accumulation. Addition of low concentrations of microparticles to a growth medium

454

containing As, such as PS at 0.04 g L-1 or PTFE at 0.1 g L-1, effectively inhibited As

455

toxicity in rice. In contrast, addition of 0.2 g L-1 PS and 0.2 g L-1 PTFE did not inhibit

456

As toxicity in rice and in fact worsened plant stress.

457

4. Conclusion

458

PS and PTFE affect transpiration and stomata of rice seedlings mainly via

459

inhibiting their root vigor , while As(III) destroys the chloroplast structure and

460

inhibits the activity of rice RuBisCo, further lowering the photosynthetic capacity of

461

rice seedlings to decreasee biomass of roots and leaves of rice seedings. During the

462

rice growing period, PS and PTFE primarily influence the rice root system, and As(III) 21

463

can inducean "oxidative burst" to rice by impairing the antioxidant enzyme structure

464

that leads to membrane lipid peroxidation and the destruction of membrane structure.

465

Due to the inhibition of microplastic particles on root activity, the ability of rice

466

seedlings to uptake As(III) was restricted, therefore, the As(III) content in tissues was

467

reduced. The effect of As(III) on rice seedlings in presence of PS was weaker than

468

that of PTFE, which was probably attributed to the higher dispersibility of PS in the

469

solution.

470

Acknowledgments

471

This work was supported by the National Natural Science Foundation of China

472

[grant numbers 41771525] and STU Scientific Research Foundation for Talents [grant

473

number NTF19025].

474

Conflicts of interest: The authors declare no conflicts of interest.

475

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593 594 27

595

Figures a

100

CK PS 0.04 PS 0.1 PTEF 0.04 PTEF 0.1

80

PS 0.2 PTEF 0.2

a

b

O2.- content (nmol g-1 FW)

de de

cd

cd

d

bc

c d

bc

bc

c

cd

*

d

d

d

d d

c

c

c

c

c

c

leaf

60

ab

ab

40 20 100 0 a

a

ab

80

60

cd

d

cd

c

cd

bc

bc c

cd cd cd

d

d

d

e de de

b

b

b

c

cd

bc

bc

c

root

596

40

20

0

1.6

4.0

3.2

As concentration (mg L-1)

597 25

b

CK PS 0.04 PS 0.1 PTEF 0.04 PTEF 0.1

20

PS 0.2 PTEF 0.2

a

ab

ab b

b

bc

bc

15

de d

d

d

cd

cd

cd d

cd

cd

cd

c

c

cd

cd

leaf

cd

c

-1

H2O2 content (µ µmol g FW)

10

bc

bc

c

c

5 0 a

20 ab

bc c

cd

10 e de

de

de

d de

cd d

d

cd

d

d

bc

c

c

c

cd

root

15

ab

b

b

d

d

de

5

0

598 599 600 601 602

1.6

3.2

4.0

-1

As concentration (mg L )

Fig. 1. Effects of Polystyrene (PS) and Polytetrafluoroethylene (PTFE) and As(III) on rice seedling (a) O2.- and (b) H2O2 (Data are mean content ± standard error (n = 3), different lowercase letters represented significant difference (P<0.05))

28

200

a

CK PS 0.04 PS 0.1 PTEF 0.04 PTEF 0.1

160

PS 0.2 PTEF 0.2

a a

a

ab

b

b

b

b

b

b

b

c

b

b

bc

bc bc

bc bc

c

cd

80

cd

cd

d

40 0 a ab

160

ab b

b bc bc bc

120

c

bc c

bc

bc bc

bc c

c

bc

c

c cd

d

c

de

80

root

FW) -1 SOD activity (U g

b

b

bc

c

leaf

120

c

cd d

de

40

0

1.6

4.0

3.2

-1 As concentration (mg L )

603 4500

a ab

a

ab

ab b

2700

-1 -1 g FW)

ab

b

ab

b

b

c

c

CAT activity (nmol min

ab

PS 0.2 PTEF 0.2 ab c

ab

b c

bc

bc c

bc

c

d

c

d

cd

1800

leaf

CK PS 0.04 PS 0.1 PTEF 0.04 PTEF 0.1

3600

900 0 a ab

1600

bc

ab

b

bc c

1200

bc

bc c

bc

c

c d

d

c

cd d

de

cd

cd d e

800

cd d

d e

de

root

b

400

0

604 605 606 607 608 609 610

0

1.6

3.2 -1 As concentration (mg L )

4.0

Fig. 2. Effects of PS or PTFE and As(III) on rice seedling (a) SOD and (b) CAT (Data are mean content ± standard error (n = 3), different lowercase letters represented significant difference (P<0.05))

29

240

CK PS 0.04 PS 0.1 PTEF 0.04 PTEF 0.1

PS 0.2 PTEF 0.2

a ab b

180 cd d

d

cd cd

cd

cd

cd

cd

cd

cd

d

60

0 a ab

240

180

cd c

cd d cd

b

bc

c

b

c

d cd

c

cd cd

c

cd

c

ab b

b c cd

b

bc

bc

root

-1 TBARS content (nmol g FW)

120

cd

d

d

bc

c

leaf

cd

cd d

b

bc

bc cd

cd

120

60

0

611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635

0

1.6

3.2

4.0

-1 As concentration (mg L ) Fig. 3. Effects of PS or PTFE and As(III) on rice TBARS (Data are mean content ± standard error (n = 3), different lowercase letters represented significant difference (P<0.05))

30

3000

a

-1 Rubisco actiity (nmol g FW)

CK PS 0.04 PS 0.1 PTEF 0.04 PTEF 0.1 2400

a a a ab ab

b bc

b b

bc bc

cd

PS 0.2 PTEF 0.2

bc c

1800

c

c c

cd

cd

de

cd

e

e

d ef

f

fg

g

1200

600

0

0

1.6

3.2

4.0

-1 As concentration (mg L )

636 1.0

b

-1 -1 Root system activity (mg g h )

CK PS 0.04 PS 0.1 PTEF 0.04 PTEF 0.1 0.8

a a

a

a a

a

bc

0.6

PS 0.2 PTEF 0.2

b

ab

bc

bc c

cd

cd

d

e

e

de

e e

e

ef

e

f

0.4

f g

fg

g

0.2

0.0

0

637 638 639 640 641 642

1.6

3.2

4.0

-1 As concentration (mg L )

Fig. 4. Effects of PS or PTFE and As(III) on rice Rubisco (a) and Root system (b) activity (Data are mean content ± standard error (n = 3), different lowercase letters represented significant difference (P<0.05))

31

100

CK PS 0.04 PS 0.1 PTEF 0.04 PTEF 0.1

80

a bc

bc cd de e

f fg

20

g

g

fg

g

de

b bc

cd

de de

e

leaf

cd

g

0 a

720 ef

fg

540

h 360

i ij

ab

bc

cd

hi

f gh h

gh

de fg

root

Arsenic content (mg kg-1)

60 40

PS 0.2 PTEF 0.2

ij j

j

ij

j

180

0

643 644 645

1.6

3.2 -1 As concentration (mg L )

4.0

Fig. 5. Effects of adding PTFE and PS on As(III) uptake of rice seedling (Data are mean content ± standard error (n = 3))

646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 32

661 662 663

664 665 666

Table Table 1 Effects of combined pollution of PS and PTFE and As(III) on rice biomass (mg pot-1), chlorophyll a, chlorophyll b and total chlorophyll (mg g-1 FW) Treatments

RB*

LB

Ca

Cb

Ct

CK

12.6±0.7a

91.0±5.1a

2.75±0.39a

1.05±0.15

3.80±0.26a

As 1.6

10.9±0.5b

84.7±7.0bc

2.50±0.28b

1.01±0.27

3.51±0.06bc

As 3.2

9.9±0.4c

81.3±5.3cd

2.30±0.19cd

1.01±0.09

3.31±0.10cd

As 4.0

9.3±0.7d

76.3±5.7e

2.18±0.14de

1.01±0.08

3.19±0.06d

PS 0.04

11.5±0.7ab

88.7±4.8ab

2.63±0.34ab

1.00±0.23

3.63±0.24b

PS 0.1

11.0±0.5ab

86.7±4.5b

2.54±0.26b

1.00±0.28

3.53±0.11bc

PS 0.2

9.9±0.6c

81.7±2.9cd

2.34±0.23c

0.96±0.06

3.30±0.18d

As 1.6+PS 0.04

11.5±0.5ab

88.3±4.5ab

2.68±0.25ab

1.03±0.19

3.71±0.14ab

As 1.6+PS 0.1

11.0±0.6ab

86.7±5.8b

2.61±0.13ab

1.01±0.18

3.62±0.22b

As 1.6+PS 0.2

9.3±0.7d

76.3±5.7e

2.37±0.22c

1.00±0.30

3.38±0.08cd

As 3.2+PS 0.04

11.0±0.7ab

86.3±2.5b

2.62±0.18ab

1.00±0.11

3.62±0.22b

As 3.2+PS 0.1

10.6±1.4bc

84.7±5.2bc

2.54±0.23b

1.01±0.09

3.55±0.29bc

As 3.2+PS 0.2

9.2±0.9d

74.0±3.7ef

2.24±0.12d

1.00±0.06

3.23±0.08d

As 4.0+PS 0.04

10.5±0.5bc

83.3±2.1c

2.53±0.15b

1.00±0.06

3.53±0.09bc

As 4.0+PS 0.1

10.0±0.6c

79.7±6.9d

2.48±0.26bc

1.00±0.19

3.48±0.14c

As 4.0+PS 0.2

9.0±1.0d

73.0±3.7f

2.05±0.14f

1.00±0.04

3.05±0.13e

PTFE 0.04

11.1±0.7ab

86.7±4.2b

2.52±0.27b

1.01±0.20

3.52±0.06bc

PTFE 0.1

10.6±0.4bc

83.0±4.1c

2.46±0.22bc

1.01±0.23

3.47±0.07c

PTFE 0.2

9.4±0.6cd

80.3±4.5d

2.22±0.11d

1.01±0.12

3.23±0.10d

As 1.6+PTFE 0.04

11.3±0.4ab

85.3±4.5bc

2.59±0.29b

1.01±0.28

3.59±0.09b

As 1.6+PTFE 0.1

10.8±0.9b

83.7±3.3c

2.52±0.25b

1.00±0.15

3.53±0.11bc

As 1.6+PTFE 0.2

9.9±0.5c

78.0±5.1de

2.30±0.20cd

1.02±0.23

3.32±0.17cd

As 3.2+PTFE 0.04

10.7±0.8b

84.0±3.7c

2.54±0.23b

1.00±0.18

3.54±0.05bc

As 3.2+PTFE 0.1

9.9±1.2c

83.0±6.2c

2.49±0.32bc

1.01±0.34

3.50±0.02bc

As 3.2+PTFE 0.2

9.6±1.0cd

77.3±3.9de

2.33±0.21c

1.00±0.14

3.33±0.15cd

As 4.0+PTFE 0.04

10.0±0.5c

82.0±7.8cd

2.51±0.34b

1.00±0.26

3.51±0.13bc

As 4.0+PTFE 0.1

9.5±1.1cd

80.3±3.4d

2.42±0.24c

1.01±0.11

3.43±0.22c

As 4.0+PTFE 0.2

9.2±0.6d

76.3±6.0e

2.11±0.10e

1.00±0.05

3.11±0.14de

*

RB means root biomass, LB means leaf biomass, Ca means chlorophyll a content, Cb means chlorophyll b content, Ct means total chlorophyll content, CK means control group. 33

667 668 669

Table 2 Effects of PS and PTFE and As(III) combined pollution on photosynthetic parameters and chlorophyll fluorescence parameters in rice

Treatments

Pn

gs

Tr

Ci

Fv/Fm

ETR

CK

13.82±1.14a

0.35±0.04a

5.94±0.65a

177.0±11.4c

0.80±0.04a

41±3.6a

As 1.6

12.89±0.45b

0.33±0.03ab

5.44±0.53c

188.7±9.9bc

0.73±0.05ab

36±4.2ab

As 3.2

11.73±0.66bc

0.29±0.04bc

5.08±0.28d

204.1±7.6b

0.70±0.05b

32±3.0b

As 4.0

10.57±0.55d

0.26±0.03c

4.61±0.43e

227.1±12.8a

0.66±0.05bc

29±5.1bc

PS 0.04

13.76±0.87a

0.34±0.02a

5.97±0.85a

174.8±15.6c

0.79±0.06a

41±4.9a

PS 0.1

13.52±0.55ab

0.33±0.04ab

5.87±0.38ab

171.2±16.7c

0.78±0.04a

39±3.2a

PS 0.2

12.59±1.10b

0.31±0.03b

5.55±0.36bc

162.2±10.1cd

0.72±0.07b

36±2.5ab

As 1.6+PS 0.04

13.55±0.47ab

0.36±0.03a

5.86±0.37ab

180.7±7.7c

0.78±0.06a

40±4.5a

As 1.6+PS 0.1

13.47±0.61ab

0.36±0.04a

5.69±0.60b

184.0±6.1bc

0.76±0.04ab

38±4.6a

As 1.6+PS 0.2

12.81±0.93b

0.34±0.03a

5.42±0.52c

190.1±9.9bc

0.72±0.04b

35±4.0ab

As 3.2+PS 0.04

13.47±0.65ab

0.34±0.04a

5.78±0.53ab

183.8±7.4bc

0.77±0.05a

38±4.2a

As 3.2+PS 0.1

13.27±0.85ab

0.32±0.03ab

5.43±0.44c

191.8±4.8bc

0.74±0.05ab

35±3.1ab

As 3.2+PS 0.2

11.89±0.69bc

0.29±0.04bc

4.97±0.41de

206.9±8.4b

0.69±0.04b

31±3.1b

As 4.0+PS 0.04

12.49±1.29b

0.32±0.03ab

5.52±0.65bc

190.6±7.5bc

0.73±0.07ab

36±3.6ab

As 4.0+PS 0.1

12.30±1.30b

0.29±0.03bc

5.14±0.43d

204.6±7.4b

0.71±0.04b

34±3.0ab

As 4.0+PS 0.2

10.63±0.65d

0.24±0.04c

4.58±0.43e

228.5±11.0a

0.64±0.03c

28±5.0bc

PTFE 0.04

13.71±0.73a

0.35±0.04a

5.90±0.37a

171.1±11.3c

0.78±0.06a

41±5.6a

PTFE 0.1

13.43±0.77ab

0.33±0.04ab

5.67±0.41b

166.4±12.7cd

0.77±0.04a

38±4.9a

PTFE 0.2

12.06±1.03bc

0.29±0.03bc

5.53±0.50bc

156.4±5.3d

0.69±0.03b

35±4.4ab

As 1.6+PTFE 0.04

13.83±0.64a

0.35±0.04a

5.73±0.34ab

181.4±10.8bc

0.77±0.04a

39±4.6a

As 1.6+PTFE 0.1

13.09±0.98ab

0.34±0.04a

5.52±0.17bc

182.5±6.5bc

0.75±0.07ab

37±2.5a

As 1.6+PTFE 0.2

12.85±0.58b

0.34±0.04a

5.45±0.32bc

188.4±10.0bc

0.73±0.03ab

36±4.5ab

As 3.2+PTFE 0.04

13.22±0.65ab

0.33±0.05ab

5.65±0.33b

185.7±8.6bc

0.76±0.05ab

37±3.1a

As 3.2+PTFE 0.1

13.06±0.49b

0.32±0.03ab

5.32±0.23c

193.2±6.4bc

0.73±0.06ab

34±6.0ab

As 3.2+PTFE 0.2

12.21±1.03b

0.31±0.05b

5.08±0.32d

200.3±8.9b

0.70±0.07b

32±3.5b

As 4.0+PTFE 0.04

12.19±0.38b

0.31±0.03b

5.35±0.36c

193.8±4.6bc

0.72±0.03b

34±4.0ab

As 4.0+PTFE 0.1

12.11±0.52bc

0.28±0.03bc

5.06±0.34d

208.7±7.8b

0.69±0.07bc

33±3.5b

As 4.0+PTFE 0.2

11.36±0.65c

0.25±0.05c

4.78±0.26e

222.4±14.9ab

0.66±0.05bc

31±4.0b

670 34

Highlights 1. Microplastic particles combined with As(III) can inhibit the growth of rice seedling. 2. Microplastic particles combined with As(III) would restrain root activity, RuBisCO activity and photosynthesis. 3. PS and PTEF decreased As(III) uptake of rice seedling.

Author Statement Zhengguo Song conceived of the idea of this study and provided financial means. Youming Dong preformed laboratory experiments. Minling Gao and Weiwen Qiu interpreted histological data and designed image analysis methods. Youming Dong and Zhengguo Song analysed the data and prepared the manuscript, all authors contributed substantially to revisions.