Department of Civil Engineering, Taiyuan University of Technology

Transcription

1 1 Supporting information for 2 3 Physiological Effects of Copper Oxide Nanoparticles and Arsenic on the Growth 4 and Life Cycle of Rice (O.sativa japonica Koshihikari ) 5 Jing Liu a*, Madie Simms a, Shuai Song b, Ryan S King a, George P Cobb a 6 a Department of Environmental Science, Baylor University 7 One Bear Place #97266, Waco, Texas, USA, b Department of Civil Engineering, Taiyuan University of Technology 9 Yingze West Street # 79, Taiyuan, Shanxi, China s: 11 * Jing Liu (Corresponding author): jing_liu@baylor.edu; 12 Madie Simms: madie_simms@baylor.edu ; 13 Shuai Song: songshuai@tyut.edu.cn 14 Ryan S King: ryan_s_king@baylor.edu 15 George P Cobb: george_cobb@baylor.edu S1

2 16 Supporting Information: 17 Number of pages: Number of figures: 6 19 Number of tables: Figure S1. Conceptual model of treatment preparation and exposure process in a 131-d exposure 22 to arsenic in soil and copper oxide nanoparticles in nutrient solution Figure S2. Light intensity record in the greenhouse (Photosynthetically Active Radiation, PAR) 24 during a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Figure S3. Temperature and humidity records in the greenhouse during a 131-d exposure to 26 arsenic in soil and copper oxide nanoparticles in nutrient solution Figure S4. SEM and TEM images from samples collected during a 131-d exposure to arsenic in 28 soil and copper oxide nanoparticles in nutrient solution. a. SEM image of copper oxide 29 nanoparticle powder; b. SEM image of rice husk outer surface of grain samples collected on day 3 131; c f. TEM images of nanoparticles and their aggregation in the solution samples collected on 31 day 14(c. As+nCuO 1 mg/l, d. ncuo1 mg/l, e. As+nCuO.1mg/L and f. ncuo.1 mg/l).9 32 Figure S5. Rice (O.sativa japonica Koshihikari ) seed germination percentages during a 131-d 33 exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution (n=2) Figure S6. Relationship between rice panicle parameters and grain numbers in treatment group 35 with or without As addition in a 131-d exposure to arsenic in soil and copper oxide nanoparticles 36 in nutrient solution (n=2) Table S1. Composition of 2% Hoagland s solution used as nutrient for the early growth stage of 39 rice seedlings during a 131-d exposure to arsenic in soil and copper oxide nanoparticles in 4 nutrient solution...12 S2

3 41 Table S2. Characterization of copper oxide nanoparticles as exposure to the rice plants in a 131-d 42 exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Table S3. Soil characteristics before and after harvesting rice plants in a 131-d exposure to 44 arsenic in soil and copper oxide nanoparticles in nutrient solution Table S4. Copper and arsenic concentration in the soil mixture before and after plant growth in a d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Table S5. Total copper concentration in solution media in a 131-d exposure to arsenic in soil and 48 copper oxide nanoparticles in nutrient solution Table S6. Total arsenic concentration in solution media in a 131-d exposure to arsenic in soil and 5 copper oxide nanoparticles in nutrient solution Table S7. 18-d seedling growth parameters in a 131-d exposure to arsenic in soil and copper 52 oxide nanoparticles in nutrient solution Table S8. 18-d seedling growth parameters in a 131-d exposure to arsenic in soil and copper 54 oxide nanoparticles in nutrient solution Table S9. Parameters of the mature rice panicles harvested from each replicate growth container 56 in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Table S1. Average grain weight and total grain weight in a 131-d exposure to arsenic in soil and 58 copper oxide nanoparticles in nutrient solution Table S11. Mature plants straw and root biomass and ratios of straw-grain and root-straw in a d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Table S 12. Accumulation of copper and arsenic in dehusked rice grains in a 131-d exposure to 62 arsenic in soil and copper oxide nanoparticles in nutrient solution Table S13. Uptake of copper and arsenic in 18-d seedlings during a 131-d exposure to arsenic in 64 soil and copper oxide nanoparticles in nutrient solution S3

4 66 Additional text is provided about fertilizer application, soil characterization and detailed 67 statistical analysis. Tabular data in this document support the depicted figures within the main 68 text of the manuscript, and may also be beneficial for those who are conducting research for 69 direct comparisons of means and ranges, or those engaged in meta-analysis of ongoing research 7 to address plant sensitivity to metals. 71 Methods 72 Whole life growth test in the greenhouse 73 Commercial substances (6% Grainger clay soil, Catalog # 2258, 4% Lowe s topsoil, # ) 74 were purchased to prepare test soils. The clay soil was ground with an automatic continuous 75 hammer mill grinder (DF-15, ECO-WORTHY). The topsoil was sifted through a 2-mm sieve. 76 Then the two types of soils were homogenized. The As treatment (1 mg/kg soil) were prepared 77 by spraying the Na 2 HAsO 4 7H 2 O (Sigma-Aldrich, lot # BCBM939V) containing solution onto 78 the spread soil surface and homogenizing (Figure S1). ncuo (Nano-Arc, 97.5%, nm, 79 APS powder, Alfa Aesar, MA, USA) treatments were prepared at 6 levels (.1, 1., 1, 5, and 8 1 mg/l) in 2% Hoagland s solution (Table S1). Hydrodynamic diameter and zeta potential of 81 ncuo in Hoagland s solution were determined with Malvern Zetasizer Nano ZS (Table 2S). 82 Twelve treatments (6 2 combinations of the ncuo and As) were prepared in total. Each 83 treatment had 2 replicate growth containers (Berry Plastics ID: T6785CP, 2.5 L) with 1.5 kg of 84 soil mixture in each. The soil in each container was saturated with deionized (DI) water. Nutrient 85 solution with appropriate ncuo concentrations were added on the first day to cover the soil with 86 4 cm (15 ml) of liquid. ncuo solutions were replenished on day 35. Ten seeds (O.sativa 87 japonica Koshihikari, Kitazawa Seed Company, CA, USA, pre-soaked for 12 hours in DI 88 water) were water-sowed in each container. Water depth was maintained with DI water. 89 Hoagland s solution was used as nutrient every other day for the first two weeks of seedling S4

5 9 growth. Thereafter, additional fertilizers (see section of Fertilizer Application) were applied to 91 optimize plant growth. 92 One day 18, seedlings were thinned to 2 about 4" apart. Seedling samples were used for 93 morphological measurement and chemical analysis. Solutions were sampled weekly during the 94 growth period. Flag leaves and grains from the same tiller (day 118 and day 131) were taken for 95 analysis at two time points. Watering was stopped when the majority of panicles started to 96 mature. Plants were harvested after one week without watering. The experiment was conducted in 97 the greenhouse for about 131 days with monitoring of the light intensity (measured with Odyssey 98 Photosynthetic Active Radiation Logger and calibrated to the PAR), humidity and temperature 99 (with the monitor of humidity and temperature) (Figure S2-S3). 1 In addition, As was mixed into the soil because: 1) As was naturally occurring in the soil, and As 11 soil contamination was worsened by all kind of human activities, especially previous arsenic- 12 containing agrochemical application. 2) the target concentration of As exposure in this study 13 approximated the concentration in soil from Texas, US. ncuo was added into the solution 14 because: 1) ncuo is not naturally occurring in appreciable amounts, but is released from 15 industrial discharge into the water system. 2) Copper-containing pesticides are usually applied as 16 suspension. 17 Fertilizer application 18 Two types of fertilizer stock solutions were made and diluted 1:1 just before use. Solution A 19 was made with 4.5 kg of Peters Water Soluble Fertilizer N.P.K in 19 L water. Solution B 11 was made with 4.5 kg of Peters Water Soluble Fertilizer N.P.K and.9 kg of Sprint in 19 L of water. Dilutions (1:1) of Solution A and B were alternatively applied into the growth 112 container on the water surface from day 49 until heading (flowering). Solution B was stopped 113 when plants flowered. S5

6 114 Because Cu is an essential element for rice plant growth, minimum Cu is needed to maintain 115 plant health. Copper concentration in Hoagland s solution is shown in Table S1, which is 116 equivalent to.95 µg/kg soil when added to the system. The estimated Cu contribution from 117 Hoagland s solution is below 48 µg/kg from one-time application of fertilizers. The Cu additions 118 from both Hoagland s solution and additional fertilizers were all below the background soil Cu 119 concentration ( Cu before in Table S1). Therefore, the influence is negligible compared to both 12 background Cu in soil and the added ncuo concentrations. 121 Soil property characterization 122 There were two types of soils before adding solutions and seeds: control soils and As containing 123 soils. Post-treatment soils were obtained after 131-d exposure of As and ncuo. Before adding 124 solutions and seeds, and after harvesting rice plants, dry soils were characterized for organic 125 matter content, phosphorus content, concentrations of exchangeable cations, cation exchange 126 capacity, ph and percent base saturation by Midwest Laboratory, Nebraska, US (Table S6). 127 Statistical analysis 128 Generalized linear models (GLMs) were used to analyze the effects of ncuo and As on the 129 growth parameters of rice plants and the interaction effect of ncuo and As on these parameters. 13 A binomial distribution was specified for the germination model, because response variables were 131 proportions but were not over dispersed 1. A gaussian distribution was used for TGW, AGW. A 132 gamma distribution was specified for other models, because response variables were continuous 133 and the distributions were right-skewed. Generalized liner mixed modeling (GLMM) was used to 134 analyze the effect of ncuo and As on the heading process with days of the experiment, because 135 GLMM accommodates specification of appropriate covariance structure to account for repeated 136 measures from the same containers over time (nested within time) and a random effect function to 137 handle the heterogeneous variances among different growth containers. Heading data were S6

7 138 averaged weekly due to the functional limit of the software. Models were assessed and accepted 139 with low heterogeneity of the residual. Analysis of variance (ANOVA) was conducted to 14 determine the main effects of ncuo and As, and their interaction effects. The p-values were 141 adjusted with Tukey method to decrease the family-wise error rate. Data were reported as the 142 mean value ± standard error of measurement (S.E.M) for each treatment group. Means were 143 considered significantly different when p <.5. Regression models were developed between 144 parameters to analyze the relationships. All statistical analyses were performed in R (version ). 146 Figure S1. Conceptual model of treatment preparation and exposure process in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution S7

8 3 25 Light intenisty / PAR /4/217 9/5/217 19/5/217 3/5/217 1/6/217 2/6/217 1/7/217 11/7/217 22/7/217 2/8/ Date 149 Figure S2. Light intensity record in the greenhouse (Photosynthetically Active Radiation, PAR) 15 during a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution /4/217 8/5/217 18/5/217 28/5/217 7/6/217 17/6/217 Temperature ( ) 27/6/217 7/7/217 Humidity 17/7/217 27/7/217 6/8/ Figure S3. Temperature and humidity records in the greenhouse during a 131-d exposure to 153 arsenic in soil and copper oxide nanoparticles in nutrient solution 154 S8

9 a b c d e f Figure S4. SEM and TEM images from samples collected during a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution. a. SEM image of copper oxide nanoparticle powder; b. SEM image of rice husk outer surface of grain samples collected on day 131; c f. TEM images of nanoparticles and their aggregation in the solution samples collected on day 14(c. As+nCuO 1 mg/l, d. ncuo1 mg/l, e. As+nCuO.1mg/L and f. ncuo.1 mg/l) 155 S9

10 Figure S5. Rice (O.sativa japonica Koshihikari ) seed germination percentages during a 131-d 158 exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution (n=2) S1

11 Figure S6. Relationship between rice panicle parameters and grain numbers in treatment group 162 with or without As addition in a 131-d exposure to arsenic in soil and copper oxide nanoparticles 163 in nutrient solution (n=2) 164 S11

12 165 Table S1. Composition of 2% Hoagland s solution used as nutrient for the early growth stage of 166 rice seedlings during a 131-d exposure to arsenic in soil and copper oxide nanoparticles in 167 nutrient solution Compound Concentration (µm) MgCl 2 4 CaCl 2 2H 2 O 2 KCl 1 H 3 BO Hoagland s Solution MnCl 2 4H 2 O 4.5 ZnSO 4 7H 2 O.5 CuSO 4 5H 2 O.15 MoO 3.7 Fe-EDTA 45 Na 2 SiO 3 5H 2 O 15 PIPES buffer PIPES (piperazine-n,n -bis(2-ethanesulfonic acid)) was used to maintain the ph ( ) of the solution S12

13 171 Table S2. Characterization of copper oxide nanoparticles as exposure to the rice plants in a d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Nominal concentration of ncuo (mg/l) Hydrodynamic diameter (nm) a Zeta potential (mv) a PDI a ± ±.69.33± ± ±.58.27± ± ±.93.15± ± ±.46.23± ± ±.5.5± The primary particle size of ncuo was nm. The measurement was done near ph 7 at 25 ºC with a 175 Malvern Zetasizer Nano when the solution was made fresh and before being dosed in the growth pot. Each 176 measurement was run in triplicate. S13

14 Table S3. Soil characteristics before and after harvesting rice plants in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Before adding ncuo containing solution Phosphorus Neutral ammonium acetate (exchangeable) Percent base saturation (computed) Soil type Organic matter % P 1 (weak bray) P2 (strong bray) OB.P 1 K Mg Ca Na ph CEC 2 (meq/1g) %K Mg% Ca% Na% Control soil 3.9 ±.4 14 ± ± ± ± ± 2754 ± 55 NA 6.8 ± ± ± ± ±.6 NA As containing soil After rice harvest Soil type variables As (mg/kg) ncuo (mg/l) 3.5 ±.2 1 ± ± ± ± ± ± ±47 7. ± ± ± ± ±.9 3.4±.4 Organic matter % P 1 (weak bray) Phosphorus Neutral ammonium acetate (exchangeable) ph P2 (strong bray) OB.P 1 K Mg Ca Na CEC 2 (meq/1g) Percent base saturation (computed) %K Mg% Ca% Na% 4.5 ±.2 77 ± ±1 96 ± ± ± ± 92 NA 7.9 ± ± ± ± ±.4 NA ±.2 74 ± ± 8 94 ± ± ± ± ± ± ±.1 9.1± ± ± ± ±.3 77 ± ± 2 82 ± ± ± ± ± ± ± ± 13.8 ± ± ± ±.1 65 ± ± 3 75 ± 2 17 ± ± ± ± ± ± ± ± ± ± ±.5 75 ± ± 8 78 ± ± ± ± ± ± ± ± ± ± ± ±.1 58 ± ± ± ± 386 ± ± ± ± 26.2 ±.2 9. ± ± ± ± ±.5 83 ± ± 1 96 ± 6 97 ± ± ± ± ± ± ± ± ± ± ±.4 92 ± ± 4 12 ± 1 97 ± ± ± ± ± ± ± ± ± ± ±.5 91 ± ± ± ± ± ± ± ±.1 25 ±.3 1. ± ± ± ± ±.6 95 ± ± 5 11 ± ± ± ± ± ± ± ± ± ± ± ±.1 86 ± ± 7 14 ± 866 ± ± ± ± ± ± ± ± ± ±. Values are means ± S.E.M, n = 2. 1 OB.P represents Olsen Bicarbonate P. 2 CEC represents Cation exchange capacity. NA: not available S14

15 Table S4. Copper and arsenic concentration in the soil mixture before and after plant growth in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Variable Cu (µg/kg) As (µg/kg) As (mg/kg) CuO (mg/l) Before After Before After 114 ± 211 Aa 123 ± 199 Aa 46 ± 48.3 Aa 374 ± 11 Aa ± 175 Aa 123 ± 2 Aa 395 ± 42.7 Aa 38 ± 119 Aa ± 112 Aa 131 ± 29 Aa 48 ± 48.2 Aa 384 ± 8.7 Aa 1 92 ± 119 Ba 126 ± 277 Aa 495 ± 163 Ba 479 ± 79.1 Ba ± 98.5 Ba 338 ± 268 Ba 55 ± 219 Ba 45 ± 23 BCa ± 263 Ba 559 ± 783 Ca 481 ± 242 Ba 417 ± 45.4 ACa 93 ± 134 Ab 13 ± 218 Ab 135 ± 414 Ab 121 ± 236 Ab.1 17 ± 489 Bb 12 ± 147 Ab 132 ± 343 Ab 114 ± 423 Ab ± 92.5 Bb 117 ± 291 Aa 133 ± 334 Ab 112 ± 157 Ab 1 18 ± 436 Bb 148 ± 854 Ba 129 ± 184 Ab 11 ± 23 Ab 5 16 ± 135 Bb 315 ± 184 Ca 134 ± 479 Ab 113 ± 29 Ab 1 11 ± 362 ABb 564 ± 545 Da 138 ± 25 Ab 12 ± 256 Ab As <.5 <.5 <.1 <.1 p-value ncuo <.1 <.1 <.1 <.1 A N 1 < <.1 Values are means ± S.E.M, n = 5 per treatment group. 1 A N = As ncuo interaction effect. A-C Means in a column at same As level with a common superscript letter are similar (p <.5). a-b Means in a column at same ncuo level with a common superscript letter are similar (p <.5). 1 A N= As ncuo interaction effect. S15

16 Table S5. Total copper concentration in solution media in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution As (mg/kg) 1 p-value ncuo Cu (µg/l) (mg/l) Day1 Day7 Day21 Day35 Day49 Day63 Day84 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 16.6 ncuo <.1 As <.1 Day <.1 N A 1 <.1 N D 2 <.1 A D 3 <.1 N A D 4 <.1 Values are means ± S.E.M, n = 5 per treatment group. 1 N A1= ncuo As interaction effect. 2 N D2 = ncuo Day interaction effect. 3 A D3= As Day interaction effect. 4 N A D4= ncuo As Day interaction effect S16

17 As (mg/kg) 1 p-value Table S6. Total arsenic concentration in solution media in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution ncuo As (µg/l) (mg/l) Day1 Day7 Day21 Day35 Day49 Day63 Day84 Day ± ± ± ± ± ± ±.8 7. ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2. ncuo.78 As <.1 Day <.1 N A 1 <.1 N D 2 <.1 A D 3 <.1 N A D 4 <.1 Values are means ± S.E.M, n = 5 per treatment group. 1 N A1= ncuo As interaction effect. 2 N D2 = ncuo Day interaction effect. 3 A D3= As Day interaction effect. 4 N A D4= ncuo As Day interaction effect. S17

18 Table S7. 18-d seedling growth parameters in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution As (mg/kg) ncuo (mg/l) Sample numbers Shoot length (cm) Root length (cm) Number of Root Branches (n = 49) 27.5 ±.62 ABa 6.3 ±.29 Aa 8.9 ±.27 Aa.1 (n = 48) 29.9 ± 1.9 ABa 5.3 ±.41 Aa 9. ±.4 ABa 1 p-value 1 (n = 47) 31.1 ±.73 Aa 8.4 ±.29 Ba 1.3 ±.38 ABCa 1 (n = 43) 29.4 ± 1.4 ABa 9.4 ±.37 Ba 1.4 ±.45 ABCa 5 (n = 42) 28.8 ±.81 ABa 9. ±.32 Ba 1.8 ±.4 Ca 1 (n = 54) 26.6 ±.86 Ba 9.4 ±.26 Ba 1.5 ±.35 BCa (n = 69) 3.9 ±.81 ABb 6. ±.27 ABa 9.5 ±.32 Aa.1 (n = 35) 31.5 ± 1. ABa 5.5 ±.42 Aa 7.6 ±.45 Bb 1 (n = 51) 32.8 ±.8 Aa 6.9 ±.33 BCb 9.7 ±.35 Aa 1 (n = 55) 28.6 ± 1. Ba 7.8 ±.28 Cb 9.6 ±.36 Aa 5 (n = 5) 28.7 ±.91 Ba 7.8 ±.42 Ca 9.2 ±.4 ACb 1 (n = 49) 31.3 ±.84 ABb 7.7 ±.39 Cb 8.2 ±.34 BCb As <.1 <.1 <.1 ncuo.1 <.1 <.1 A N <.1 Values are means ± S.E.M. A-C Means in a column at same As level with a common superscript letter are similar (p <.5). a-b Means in a column at same ncuo level with a common superscript letter are similar (p <.5). 1 A N= As ncuo interaction effect. S18

19 Table S8. 18-d seedling growth parameters in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution As (mg/kg) ncuo (mg/l) Sample numbers Shoot dry weight (mg) Root dry weight (mg) (n = 53) 25.1 ±.91 Aa 3.2 ±.19 Aa.1 (n = 48) 25.1 ± 1.18 Aa 2.4 ±.19 Ba 1 p-value 1 (n = 47) 32.6 ± 1.29 Ba 4.2 ±.22 Ca 1 (n = 43) 3. ± 1.39 Ba 5.6 ±.27 Da 5 (n = 45) 3.7 ± 1.29 Ba 6.1 ±.28 Da 1 (n = 47) 31. ± 1.29 Ba 6.5 ±.25 Da (n = 8) 27.7 ±.87 Aa 3.4 ±.17 Aa.1 (n = 45) 23.8 ± 1.7 Ba 2.3 ±.15 Ba 1 (n = 5) 3.5 ± 1.26 Aa 3.6 ±.23 Ab 1 (n = 5) 28.1 ± 1.14 Aa 3.6 ±.17 Ab 5 (n = 47) 26.2 ± 1.7 ABb 3.4 ±.16 Ab 1 (n = 45) 26.9 ±.85 ABb 3.1 ±.15 Ab As <.1 <.1 ncuo.18 <.1 A N 1.9 <.1 Values are means ± S.E.M. A-D Means in a column at same As level with a common superscript letter are similar (p <.5). a-b Means in a column at same ncuo level with a common superscript letter are similar (p <.5). 1 A N= As ncuo interaction effect. S19

20 Table S9. Parameters of the mature rice panicles harvested from each replicate growth container in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution As (mg/kg) 1 p-value CuO (mg/l) TNRP PAL PBN SBN SN PW TPW 11.6 ±.43 ABa 14.9 ±.12 ABa 6. ±.9 ABa 7.5 ±.16 ABa 52. ±.87 ABa 1. ±.4 Aa 11.7 ±.4 ABa ±.52 Aa 15.9 ±.25 Aa 6.3 ±.12 Aa 8.2 ±.36 Aa 56. ± 1.6 Aa 1. ±.3 Aa 1.3 ±.45 Aa ±.62 BCDa 14.7 ±.23 Ba 5.9 ±.12 ABa 7.1 ±.34 ABa 49. ± 1.52 Ba 1. ±.3 Aa 13.1 ±.55 BCa ±.44 BCa 14.5 ±.16 Ba 6. ±.8 ABa 7.2 ±.26 ABa 5.1 ± 1.13 Ba 1.1 ±.2 Aa 13.6 ±.48 Ca ±.56 CDa 14.5 ±.14 Ba 5.9 ±.8 ABa 6.9 ±.22 Ba 49.1 ±.94 Ba 1. ±.2 Aa 14.4 ±.45 Ca ±.63 Da 14.4 ±.37 Ba 5.8 ±.1 Ba 6.7 ±.27 Ba 46.8 ± 1.18 Ba 1. ±.3 Aa 15.1 ±.37 Ca 11.9 ±.45 Aa 15.5 ±.28 Aa 6.2 ±.1 ABa 8.4 ±.35 ABa 54.7 ± 1.64 Aa 1.2 ±.4 Ab 13.7 ±.48 Ab ±.42 Ab 15.8 ±.19 Aa 6.2 ±.9 ABa 8.5 ±.37 ABa 56. ± 1.77 ABa 1.3 ±.5 ABb 15.8 ±.47 Bb ±.56 Aa 15.9 ±.39 Ab 6.2 ±.13 ABa 8.2 ±.33 ABCb 53.7 ± 1.53 Ab 1.2 ±.4 ABb 15. ±.6 ABb ±.44 Aa 15.4 ±.19 Ab 6. ±.9 ABa 8. ±.26 ACa 52.5 ± 1.21 Aa 1.1 ±.3 Aa 13.2 ±.41 Aa ±.49 Ab 15.6 ±.21 Ab 5.8 ±.11 Aa 7. ±.41 Ca 5.7 ± 1.4 Aa 1.1 ±.3 Ab 13.4 ±.48 Aa ±.63 Ab 17. ±.36 Bb 6.5 ±.15 Bb 9.5 ±.49 Bb 62.4 ± 2.37 Bb 1.3 ±.5 Bb 14.7 ±.38 ABa As <.5 <.1 <.1 <.1 <.1 <.1 <.1 ncuo <.1 <.1 <.5 <.1 <.1.12 <.1 A N 1 <.1 <.1 <.1 <.1 <.1 <.1 <.1 TNRP: Total number of ripe panicles. PAL: panicle axis length (cm). PBN: primary branch number. SBN: secondary branch number per panicle. SN: total spikelet numbers per panicle. PW: average panicle weight (g). TPW: total panicle weight in each replicate container (g). Values are means ± S.E.M, n = 2 per treatment group. A-D Means in a column at the same As level with a common superscript letter are similar (p <.5). a-b Means in a column at the same ncuo level with a common superscript letter are similar (p <.5). 1 A N = As ncuo interaction effect. S2

21 Table S1. Average grain weight and total grain weight in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution As (mg/kg) ncuo (mg/l) Total grain weight (g) Average grain weight (mg) 1.6 ±.46 ABa 24.1 ±.15ABa 1 p-value.1 9. ±.45 Aa 23.6 ±.15Aa ±.5 BCa 24.7 ±.19 BCa ±.44 CDa 25.2 ±.16 Ca ±.44 CDa 24. ±.11 ABa ±.38 Da 23.5 ±.29 Aa 12. ±.46 Ab 25.3 ±.22 ABb ±.45 Bb 25.9 ±.27 ACb ±.48 ABb 26.2 ±.19 Cb ±.41 Aa 23.2 ±.19 Db ±.41 Aa 24.7 ±.28 Bb ±.31 ABa 25. ±.19 Bb As <.1 <.1 ncuo.1 <.1 A N 1 <.1 <.1 Values are means ± S.E.M. n=2 per treatment for total grain weight. n = 5 per treatment for average grain weight. A-D Means in a column at same As level with a common superscript letter are similar (p <.5). a-b Means in a column at same ncuo level with a common superscript letter are similar (p <.5). 1A N= As ncuo interaction effect. S21

22 Table S11. Mature plants straw and root biomass and ratios of straw-grain and root-straw in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution As (mg/kg) ncuo (mg/l) Straw DW (g) Grain to straw ratio Rice root DW (g) Root to straw ratio 21.2 ± 1.56 ABa.52 ±.3 Aa 1.7 ±.5 ABa.8 ±.7 ABa ± 1.78 Ca.35 ±.23 Ba 1.8 ±.12 ABa.7 ±.1 Aa 1 p-value ± 1.6 ADa.53 ±.26 Aa 2.3 ±.12 ACa.1 ±.7 Ba ± 1.28 Ba.7 ±.27 Ca 1.6 ±.11 Ba.9 ±.9 ABa ±.64 Ca.49 ±.19 Aa 2.8 ±.18 Ca.11 ±.9 Ba ±.57 CDa.53 ±.19 Aa 2.5 ±.15 Ca.1 ±.6 ABa 2.8 ± 1.8 ABa.59 ±.23 ABa 1.9 ±.21 Aa.9 ±.11 Aa ±.74 ACb.64 ±.26 ABb 2. ±.13 Aa.9 ±.3 Aa ±.83 ABa.69 ±.28 Ab 1.7 ±.12 ABb.8 ±.3 Ab ± 1.38 ABa.64 ±.22 ABa 1.6 ±.13 ABa.9 ±.5 Aa ±.73 Bb.63 ±.2 ABb 1.4 ±.1 Bb.7 ±.4 Ab ±.81 Ca.53 ±.16 Ba 2. ±.16 Ab.8 ±.9 Aa As <.1 <.1 < ncuo <.1 <.1 <.1.79 A N 1 <.1 <.1 <.1.4 Values are means ± S.E.M, n = 2 per treatment group for straw DW, n =5 per treatment group for rice root DW. A-C Means in a column at the same As concentration with a common superscript letter are similar (p <.5). a-b Means in a column at the same ncuo concentration with a common superscript letter are similar (p <.5). 1 A N = As ncuo interaction effect. S22

23 Table S 12. Accumulation of copper and arsenic in dehusked rice grains in a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Variable Cu (ng/g) As (ng/g) As (mg/kg) CuO (mg/l) Dehusked grain Dehusked grain 989 ± 471 Aa 44.4 ± 4.7 Aa ± 577 ABa 39.2 ± 4.33 Aa ± 292 Ba 4.9 ± 6.32 Aa 1 95 ± 438 ABa 48.1 ± 4.41 Aa ± 319 ABa 49. ± 4.75 Aa ± 211 ABa 38.5 ± 1.75 Aa 682 ± 4 Ab 198 ± 21.1 Ab ± 197 ABb 132 ± 8.25 ABb ± 477 ABa 138 ± 7.5 ABb ± 544 BCa 167 ± 15.9 ABb p-value 5 11 ± 466 Cb 128 ± 15.4 Bb ± 415 BCa 175 ± 13.7 ABb As.5 <.1 ncuo.9.25 A N 1 <.1.1 Values are means ± S.E.M, n = 5 per treatment group. A-C Means in a column at same As level with a common superscript letter are similar (p <.5). a-b Means in a column at same ncuo level with a common superscript letter are similar (p <.5). 1 A N= As ncuo interaction effect S23

24 Table S13. Uptake of copper and arsenic in 18-d seedlings during a 131-d exposure to arsenic in soil and copper oxide nanoparticles in nutrient solution Variable Cu (ng/g) As (ng/g) As ncuo (mg/kg) (mg L) Root Shoot Root Shoot 212 ± 259 Aa 128 ± 115 Aa 192 ± 362 Aa 612 ± 7.3 ABa ± 199 Aa 114 ± 486 Aa 443 ± 652 Ba 714 ± 41.2 Aa 1 21 ± 517 ABa 11 ± 15 Aa 31 ± 599 ABa 511 ± 21.3 Ba 1 16 ± 114 Ba 962 ± 674 Aa 279 ± 278 ABa 585 ± 49.7 ABa 5 76 ± 464 Ca 173 ± 9.7 Ba 259 ± 176 ABa 532 ± 17.6 Ba ± 675 Ca 183 ± 58 Ba 219 ± 147 Aa 543 ± 17.4 Ba 16 ± 535 Ab 868 ± 133 Ab 212 ± 263 Ab 285 ± 63.3 Ab ± 142 Ab 843 ± 244 ABa 178 ± 273 Ab 298 ± 81.3 Ab ± 124 ABb 878 ± 23 ABa 16 ± 175 Ab 324 ± 175 Ab ± 691 Ba 12 ± 159 Bb 164 ± 225 Ab 39 ± 187 Ab ± 179 Ca 166 ± 34 Cb 811 ± 643 Bb 317 ± 131 Ab 1 13 ± 449 Db 28 ± 525 Db 27 ± 251 Ab 36 ± 13 Ab As.28 <.1 <.1 <.1 p-value ncuo <.1 <.1 < A N 1 <.1 <.1 <.1 <.1 Values are means ± S.E.M, n = 5 per treatment group. A-D Means in a column at same As level with a common superscript letter are similar (p <.5). a-b Means in a column at same ncuo level with a common superscript letter are similar (p <.5). 1 A N= As ncuo interaction effect S24

25 References 1. Crawley, M. J., The R book. 2nd ed.; Wiley: Chichester, West Sussex, United Kingdom, 213. S25