Accepted Manuscript Residual effects of monoammonium phosphate, gypsum and elemental sulfur on cadmium phytoavailability and translocation from soil to wheat in an effluent irrigated field Muhammad Farooq Qayyum, Muhammad Zia ur Rehman, Shafaqat Ali, Muhammad Rizwan, Asif Naeem, Muhammad Aamer Maqsood, Hinnan Khalid, Jörg Rinklebe, Yong Sik Ok PII: S0045-6535(17)30175-3 DOI: 10.1016/j.chemosphere.2017.02.006 Reference: CHEM 18769 To appear in: Chemosphere Received Date: 28 November 2016 Revised Date: 23 January 2017 Accepted Date: 01 February 2017 Please cite this article as: Muhammad Farooq Qayyum, Muhammad Zia ur Rehman, Shafaqat Ali, Muhammad Rizwan, Asif Naeem, Muhammad Aamer Maqsood, Hinnan Khalid, Jörg Rinklebe, Yong Sik Ok, Residual effects of monoammonium phosphate, gypsum and elemental sulfur on cadmium phytoavailability and translocation from soil to wheat in an effluent irrigated field, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. 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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. ACCEPTED MANUSCRIPT Highlights  Residual monoammonium phosphate (MAP) and gypsum reduced the Cd uptake in wheat  Amendment of residual elemental sulfur (S) increased Cd uptake in plants  Gypsum had the highest cost-benefit ratio compared with MAP and elemental S  Gypsum may be used to enhance crop production in Cd-contaminated soils ACCEPTED MANUSCRIPT 1 Residual effects of monoammonium phosphate, gypsum and elemental sulfur on cadmium 2 phytoavailability and translocation from soil to wheat in an effluent irrigated field 3 4 Muhammad Farooq Qayyum1, Muhammad Zia ur Rehman2, Shafaqat Ali3, Muhammad 5 Rizwan3*, Asif Naeem4, Muhammad Aamer Maqsood2, Hinnan Khalid2, Jörg Rinklebe5, 6 Yong Sik Ok6 7 1Department 8 Zakariya University, Multan, Pakistan 9 2Institute 10 Pakistan 11 3Department 12 Allama Iqbal Road, 38000 Faisalabad, Pakistan 13 4Nuclear 14 Pakistan 15 5University 16 School of Architecture and Civil Engineering, Soil- and Groundwater-Management, 17 Pauluskirchstraße 7, 42285 Wuppertal, Germany 18 6 19 Kangwon National University, Chuncheon 200-701, Korea 20 *Corresponding Author: mrazi1532@yahoo.com 21 Abstract 22 Cadmium (Cd) accumulation in agricultural soils is one of the major threats to food security. 23 The application of inorganic amendments such as mono-ammonium phosphate (MAP), gypsum of Soil Science, Faculty of Agricultural Sciences & Technology, Bahauddin of Soil and Environmental Sciences, University of Agriculture, Faisalabad 38040, of Environmental Sciences and Engineering, Government College University, Institute for Agriculture and Biology (NIAB), P.O. Box 128, Jhang Road, Faisalabad, of Wuppertal, Institute of Foundation Engineering, Water- and Waste-Management Korea Biochar Research Center and School of Natural Resources and Environmental Science, 1 ACCEPTED MANUSCRIPT 24 and elemental sulfur (S) could alleviate the negative effects of Cd in crops. However, their long- 25 term residual effects on decreasing Cd uptake in latter crops remain unclear. A field that had 26 previously been applied with treatments including control and 0.2, 0.4 and 0.8% by weight of 27 each MAP, gypsum and S, and grown with wheat and rice and thereafter wheat in the rotation 28 was selected for this study. Wheat (Triticum aestivum L.) was grown in the same field as the 29 third crop without further application of amendments to evaluate the residual effects of the 30 amendments on Cd uptake by wheat. Plants were harvested at maturity and grain, and straw 31 yield along with Cd concentration in soil, straw, and grains was determined. The addition of 32 MAP and gypsum significantly increased wheat growth and yield and decreased Cd 33 accumulation in straw and grains compared to control while the reverse was found in S 34 application. Both MAP and gypsum decreased AB-DTPA extractable Cd in soil while S 35 increased the bioavailable Cd in soil. Both MAP and gypsum increased the Cd immobilization 36 in the soil and S decreased Cd immobilization in a dose-additive manner. We conclude that 37 MAP and gypsum had a significant residual effect on decreasing Cd uptake in wheat. The cost- 38 benefit ratio revealed that gypsum is an effective amendment for decreasing Cd concentration in 39 plants. 40 41 Keywords: Cadmium immobilization, Field experiment, Elemental sulfur, Gypsum, Mono- 42 ammonium phosphate, Residual effect 43 44 45 46 2 ACCEPTED MANUSCRIPT 47 1. Introduction 48 Soil is both a sink for contaminants including toxic heavy metals such as cadmium (Cd), and a 49 source of nutrients for supporting plant growth and development (Rehman et al., 2015; 50 Mohiuddin et al., 2016; Rizwan et al., 2016a). In numerous plant growth trials, it is evident that 51 heavy metals significantly influence the plant growth as well as cause health hazards (Rizwan et 52 al., 2012; Adrees et al., 2015a; Tauqeer et al., 2016; Wang et al., 2016). Compared to other toxic 53 heavy metals, Cd is readily mobile and active metal and is toxic even at lower concentration (He 54 et al., 2015; Rizwan et al., 2016a; Ran et al., 2016). Recent studies revealed that Cd could be the 55 key factor behind unavailability of safe food on a sustainable basis (Ran et al., 2016; Yang et al., 56 2016). Cadmium toxicity is also harmful to plant growth and development, as it hinders plant’s 57 physiological and metabolic processes (Li and Xu, 2015; Lopez-Luna et al., 2016). 58 The agricultural sector is a vital component of the Pakistani economy, and water scarcity is a 59 continuing concern for overall productivity due to higher dependency on single river system 60 (Rehman et al., 2015; Ngigi, 2016). Thus, unavailability of fresh water forces the farmers to use 61 raw city effluents and sewage water as an alternate source of irrigation (Khan et al., 2016a; 62 Rehman et al., 2015). Soils irrigated with raw city effluents might be a potential source of Cd 63 which may be taken up by plants causing toxicities at different levels. Thus, there is an urgent 64 need to employ remediation techniques aiming to reduce Cd availability and uptake by plants. 65 Different remediation techniques may be used for the reduction of Cd availability and uptake by 66 plants (Adrees et al. 2015b; Sabir et al., 2015). The use of inorganic amendments for Cd 67 decontamination is more feasible and economic strategy when compared with other remediation 68 strategies especially dealing with large areas (Rizwan et al., 2016b). Complexation and 69 precipitation of Cd by the use of organic or inorganic amendments are documented methods for 3 ACCEPTED MANUSCRIPT 70 reducing Cd uptake by plants (Li et al., 2016; Rizwan et al., 2016b). Inorganic amendments like 71 gypsum, phosphate fertilizers, limestone, elemental sulfur, and iron oxides have proved to be 72 suitable amendments for immobilization of Cd in the soil and reducing its uptake by plants (Cui 73 et al., 2016; Zhao et al., 2016). In contrast, there is also a chance that Cd availability may 74 increase by the application of certain inorganic material like sulfur (Asgher et al., 2014; Khan et 75 al., 2015). In a previous study, we reported that the application of gypsum and mono-ammonium 76 phosphate decreased the Cd uptake by wheat and rice crops while elemental sulfur increased the 77 Cd uptake in these crops grown under field conditions (Rehman et al., 2015). However, little is 78 known about the residual effects of these amendments on Cd uptake by plants. Therefore, 79 studying the residual effects of these amendments on subsequent crops may provide a better 80 understanding of the suitability of these amendments for in situ Cd immobilization in the soil. 81 Our objective was to investigate the residual effects of gypsum, mono-ammonium phosphate 82 (MAP) and elemental sulfur for in situ Cd immobilization in the soil and uptake by wheat plants 83 grown in an aged-contaminated soil receiving raw city effluents for 30 years. 84 2. Materials and methods 87 2.1. Site selection and characteristics 88 A field was selected in the suburbs of Multan city (30° 12′ N, 71° 28′ E and 215 m above sea 89 level) of Punjab, Pakistan. The selected field had been previously irrigated with raw city 90 effluents and sewage water for last 30 years. The field used was from our earlier experiment 91 (Rehman et al., 2015) where amendments were applied in ten treatments viz: control (T1), MAP 92 @ 0.2% (T2), MAP @ 0.4% (T3), MAP @ 0.8 % (T4), gypsum @ 0.2% (T5), gypsum @ 0.4 % 85 86 4 ACCEPTED MANUSCRIPT 93 (T6), gypsum @ 0.8 % (T7), elemental sulfur @ 0.2 % (T8), elemental sulfur @ 0.4 % (T9) and 94 elemental sulfur @ 0.8 % (T10) with four replicates. No further amendments were applied in the 95 present study. Prior to the sowing of wheat, a representative soil sample was collected from 96 control plots and analyzed for various physicochemical properties as follows: 97 Jenway pH meter (Model 671P) was used for pH determination of saturated soil paste (pH) by 98 directly pushing the electrode into the saturated soil paste. The ammonium bicarbonate 99 diethylene-triamine-penta-acetic acid (AB-DTPA) extraction method was exploited for 100 determination of bioavailable heavy metals (Soltanpour, 1985). For this, air-dried soil (10.0 g) 101 was added in 20 ml of freshly prepared AB-DTPA solution and the mixture was horizontally 102 shaken for about 30 min and then filtered for the determination of bioavailable heavy metals by 103 using an atomic absorption spectrometer (Solar S-100; Thermo Electron, Massachusetts, USA). 104 Amacher (1996) procedure was followed for the determination of total metal concentration in 105 soil. In brief, 10 ml of concentrated HNO3 were added in air-dried soil (1.0 g) and kept overnight 106 and then the mixture was heated at 200 °C. After cooling the mixture, 1 ml of HNO3 and 4 ml of 107 HClO4 were added and again heated to 200 °C until fumes of HClO4 appeared, cooled and added 108 1:10 HCl and heated at 70 °C for 1 h and finally 50 ml volume was made with 1 % HCl and 109 filtered (Whatman filter paper No. 42) the mixture for further analysis. Soil texture was 110 determined following the protocol of Bouyoucos, (1962). Soluble ions including Ca2+, Mg2+, 111 Na+, K+, Cl-, CO32-, HCO3-, and SO42- were determined by the titration method (Richards, 112 1954). Electrical conductivity of the saturation extract (ECe), sodium adsorption ratio (SAR) and 113 cation exchange capacity (CEC) were also determined following the methods described by 114 Richards (1954). Calcium carbonate contents were determined by calcimeter method (Moodie 5 ACCEPTED MANUSCRIPT 115 et al., 1959) while soil organic matter (OM) was determined following Walkley-Black method 116 (Jackson, 1962). 117 The soil was a sandy clay loam and physicochemical properties of the soil are given in Table 1. 118 Available Cd values vary in the pots with the amendments applied (Rehman et al., 2015). Raw 119 city effluent that was used for irrigation was analyzed before each irrigation and the average 120 properties of the water along with permissible limits are also given in Table 1. 121 122 2.2. Sowing of wheat 123 Wheat variety Inqalab-91 was sown by a broadcast method using a seed rate 125 kg ha-1 in 124 rotation after rice of the previous experiment (Rehman et al., 2015) which had been followed by 125 first wheat crop grown with amendment application and the rice without application of 126 amendments. Fertilizers NPK @ 30-75-20 kg ha-1, were applied as urea, diammonium 127 phosphate and sulfate of Potash at the time of sowing, respectively. Five irrigations (each of 4 128 inch depth) with raw city effluents were applied to mature the crop, while 67.3 mm rainfall was 129 recorded during the growth period (Agricultural Metrology Cell, CCRI, Multan, Pakistan). The 130 experimental design was a completely randomized block design (RCBD). 131 132 2.3. Plant sampling and analysis 133 The wheat was harvested at maturity, after 145 days of sowing, and plant samples were collected 134 after the separation of grain and straw. Plant samples were washed with tap water and then 135 gently rinsed in 1 % HCl and then with distilled water. Samples were oven dried at 70 °C till 136 constant weight. Willey mill having stainless steel blades was used for grinding of samples. For 137 digestion, 1.0 g straw/grain sample was taken in 10 ml di-acid mixture (concentrated nitric acid 6 ACCEPTED MANUSCRIPT 138 and perchloric acid in a ratio of 3:1) and left overnight. Thereafter, samples were heated on a hot 139 plate till clear solution appeared. Digested samples were diluted up to 25 ml using de-ionized 140 water (Ryan et al., 2001). Atomic absorption spectrometer was used for Cd quantification in 141 digested plant samples (Solar S-100; Thermo Electron, Massachusetts, USA). 142 Total Cd uptake by wheat plants was calculated by multiplying the dry biomass to the Cd 143 concentrations in shoots or roots. 144 Total Cd uptake = Cd concentration in shoots or roots × shoot or root dry weight 145 Shoots to grain translocation of Cd was assessed by translocation index (TI) by using the 146 following equation TI (%) = 147 Cd (grains) × 100 Cd (grains + straw) 148 Moreover, by modifying this formula we calculated Cd harvest index by a wheat crop as 149 follows: 150 Cd Harvest Index = (CdG + CdS) × 100 (CdG + CdGS + CdSS) 151 152 Where CdG, CdS and CdSS represent Cd content in grains, straw and soil, respectively. 153 For economic analysis, benefit cost ratio (BCR) was calculated following the method described 154 by Blank and Tarquin (1998) as follows: BCR = 155 156 B C Where B and C, represent the benefits and cost of wheat production, respectively. 157 158 2.4. Soil sampling and analyses 7 ACCEPTED MANUSCRIPT 159 After harvest of wheat, soil samples from each experimental unit were collected at depths (0–15 160 and 15–30 cm) separately. The soil samples were air-dried, ground with wooden roller, passed 161 through 2-mm sieve and stored in plastic jars. Soil pH and AB-DTPA extractable Cd were 162 determined as described above (Section 2.1). 163 Cadmium immobilization was calculated by the method of Lee et al. (2013) as follows: 164 Immobilized Cd (%) = (Extractable Cd control ‒ Extractable Cd in sample × 100 Extractable Cd in control 165 2.5. Statistical analysis 166 All the data were statistically analyzed using one-way ANOVA at a significance level of P ≤ 167 0.05 with SPSS 21.0 software for Windows. Single-step multiple comparisons of means were 168 performed via Tukey’s-HSD post hoc test. Different letters on the histograms indicate that the 169 means are statistically different at P ≤ 0.05 levels. The data presented are means of four 170 replicates as described above. 171 172 3. Results 173 3.1. Grain and straw yield 174 The yield of wheat grain was significantly (p < 0.05) affected by most treatments (Table 2), 175 however, application of elemental sulfur @ 0.2 % (T8) and 0.4 % (T9) did not affect the grain 176 yield. On the other hand, application of elemental sulfur @ 0.8 % (T10) decreased the grain yield 177 by 14% compared to the control. Except for elemental sulfur, all other amendments increased the 178 yield of grain while the maximum increment (4.18 Mg ha-1) was recorded when gypsum was 179 applied @ 0.8 % (T7) compared to the control. The applied elemental sulfur @ 0.8 % (T10) did 180 not affect the yield of straw while all other treatments significantly (p < 0.05) increased the yield 181 of straw compared to the control (Table 2). The maximum straw yield (6.02 Mg ha-1) was 8 ACCEPTED MANUSCRIPT 182 recorded with gypsum @ 0.8 % (T7) while the decreasing order of treatment efficiency was 183 observed as T7 > T6 > T8 > T3 > T4 > T5 > T9 > T2 > T1 > T10 for increasing the straw yield. 184 Moreover, a negative correlation was observed between post-harvest soil pH and grain and straw 185 Cd concentrations (Figure 2). 186 187 3.2. Cadmium concentration and uptake by wheat 188 Cadmium concentration in wheat was significantly (p < 0.05) affected by all amendments (Table 189 2). Elemental sulfur @ 0.2 % (T8) did not show any effect on straw Cd concentration. Maximum 190 Cd concentration in grains (0.13 mg kg-1) and straw (0.26 mg kg-1) was recorded with the 191 application of elemental sulfur @ 0.8 % (T10) while all other amendments reduced the Cd 192 concentration both in grains and straw compared to the control. 193 Elemental sulfur @ 0.2% (T8) did not affect Cd uptake by wheat grain while elemental sulfur @ 194 0.4 % (T9) and @ 0.8 % (T10) increased Cd uptake by 13% and 45% compared to the control 195 respectively (Table 2). Elemental sulfur @ 0.2 % (T8), 0.4 % (T9) and 0.8 % (T10) increased the 196 Cd uptake by 10%, 15%, and 66% in straw respectively while a decreasing trend was observed 197 for Cd uptake by wheat straw with all other treatments. Both gypsum and MAP significantly 198 decreased the Cd uptake by wheat straw and grains compared to the control in a dose-additive 199 manner. 200 201 3.3. Post-Harvest soil Cd and pH 202 Post-harvest AB-DTPA extractable soil Cd revealed significant (p < 0.05) effects of treatments 203 (Table 3). The application of elemental sulfur @ 0.2% (T8) and gypsum @ 0.2% (T5), did not 204 affect Cd concentration in soil at both the depths, i.e. 0-15 cm and 15-30 cm. Whereas, the higher 9 ACCEPTED MANUSCRIPT 205 application rate of elemental sulfur @ 0.4 % (T9) and 0o.8% (T10) significantly increased the Cd 206 concentration which was about 8% and 62% higher in 0-15 cm soil depth respectively compared 207 to the control. In 15-30 cm soil depth, Cd concentration increased by 13% and 15% with 208 elemental sulfur @ 0.4% (T9) and @ 0.8% (T10) respectively. In contrast, the other treatments 209 significantly reduced the Cd concentration in both soil depths compared to control. 210 The soil pH was significantly affected by the application of most of the treatments (Table 3). 211 There was no change in pH of 0-15 cm soil with gypsum @ 0.2% (T5) compared to the control. 212 The maximum increase in the soil pH for 0-15 cm (1.02%) was recorded with gypsum @ 0.4% 213 (T6) and for 15-30 cm (1.28%) with gypsum @ 0.2% (T5). Elemental sulfur decreased the pH of 214 soil in both depths at all application rates while MAP and gypsum did not affect the soil pH at 215 any depth. Treatment efficiency in pH decreasing order was observed as: T6 > T3 > T7 > T2 > T4 216 > T1 = T5 > T8 > T9 > T10 and T5 > T4 = T3 = T2 > T6 > T7 > T1 > T8 > T10 > T9 for 0-15 cm and 217 15-30 cm, respectively. Moreover, a negative correlation was observed between post-harvest soil 218 pH and AB-DTPA extractable soil Cd concentrations (Figure 2). 219 220 3.4. Cadmium translocation index and harvest index 221 Significant (p < 0.05) differences were observed for Cd translocation from shoot to grains 222 (Figure 1A). As compared to the control, elemental sulfur decreased the Cd shoot to grain 223 transfer by 0.05%, 0.01% and 0.083% for 0.2%, 0.4% and 0.8% treatments respectively. Both 224 MAP and gypsum increased the shoot to grain Cd translocation index compared to the control. 225 Maximum and minimum shoot to grain Cd translocation index was observed with gypsum @ 0.8 226 % (T7) and elemental sulfur @ 0.8 % (T10) respectively. 10 ACCEPTED MANUSCRIPT 227 Cadmium harvest index calculation is a proximal approach. It explains the Cd harvest or removal 228 from the soil by the use of plants such as wheat crop in the present study. Significant (p < 0.05) 229 changes were detected in Cd harvest index with the application of all amendments (Figure 1B). 230 Results showed that all levels of gypsum (0.2%, 0.4%, and 0.8%) significantly decreased the Cd 231 harvest index. Other two amendments, MAP, and sulfur, showed a considerable increase in the 232 Cd harvest index at all levels of the amendments applied. Maximum Cd harvest index (0.376) 233 was observed with MAP @ 0.4% (T3) while the minimum Cd harvest index (0.189) was 234 observed with gypsum @ 0.4% (T6). 235 236 3.5. Cadmium immobilization in post-wheat harvest soil 237 All treatments significantly (p < 0.05) affected the Cd immobilization in the post-harvest soil at 238 both depths (Figure 1C. Cadmium immobilization in soil increased with the increasing doses of 239 MAP and gypsum but decreased with increasing sulfur doses. Maximum Cd immobilization for 240 0-15 cm (0.91) was observed with MAP @ 0.8 % (T4) and for 15-30 cm (0.21) with MAP @ 241 0.4% (T3) while minimum Cd immobilization for 0-15 cm (-0.555) and for 15-30 cm (-0.154) 242 was found with elemental sulfur @ 0.8 % (T10). 243 244 3.6. Economics of the treatments 245 The cost-benefit ratio for three consecutive years was calculated to determine the most efficient 246 as well as economic treatment for reduction in Cd uptake by wheat plants. The maximum cost- 247 benefit ratio was detected with gypsum amendment for the three levels (0.2%, 0.4% and 0.8%) 248 being the highest for 0.2% level while the minimum was obtained with sulfur amendments 249 (Figure 3). Among three years, the cost-benefit ratio of the 3rd year showed more significant 11 ACCEPTED MANUSCRIPT 250 results compared to the previous two years. This was due to residual effects of amendments 251 applied in the first year and no use for the following two years of crop rotation. 252 253 4. Discussion 254 Inorganic amendments were incorporated in the Cd-contaminated soil in a previous experiment 255 (Rehman et al., 2015) on wheat and rice and here, there residual effects on Cd uptake and 256 accumulation in wheat were studied. The results demonstrated that gypsum and MAP increased 257 the straw and grain yield compared to the control in a dose-additive manner (Table 2). The 258 increase in grain and straw yield due to these amendments might be due to the significant role of 259 calcium and phosphorus for growth and yield enhancement (Kim et al., 2016). Within this 260 context, it was reported that Ca and P have a high potential to immobilize Cd in the soil (Sun et 261 al., 2016; Wu et al., 2016). The immobilization of Cd in the sewage-irrigated soil may be another 262 reason of higher straw and grain yields. Several studies revealed that Cd toxicity inhibits the 263 plant growth and development by causing severe disorders in plant physiological and metabolic 264 processes such as photosynthesis, respiration, chlorophyll content, nitrogen metabolism, stomatal 265 conductance, and nutrient imbalance (Adrees et al., 2015b; Lopez-Luna et al., 2016; Rizwan et 266 al., 2016b). Cadmium being a cation also competes with essential nutrients during uptake by 267 plants and causes Cd toxicity in plants as well as deficiency of other nutrients (Rizwan et al., 268 2016c). 269 In contrast to the positive effects of gypsum and MAP, sulfur mobilized the seed in soil and thus 270 lower grain and straw yield was obtained along with the highest rate of a sulfur application 271 (Table 2). Khan et al. (2015) also reported that sulfur has the potential to enhance Cd mobility 272 under wheat growth. Similarly, sulfur application at the highest rate (0.8 %) resulted in the 12 ACCEPTED MANUSCRIPT 273 maximum Cd concentration in both grains and wheat straw (Table 2). The application of sulfur is 274 able to support the plant growth and the accumulation of sulfate into plant tissues may enhance 275 the tolerance of plants against stresses like Cd toxicity (Khan et al., 2016b; Saifullah et al., 276 2016). However, sulfur amendment is also able to increase the Cd solubility which results in 277 higher Cd accumulation both in grain and straw of wheat (Rehman et al., 2015). Similarly, sulfur 278 application increased the Cd concentrations in maize shoots (Cui and Wang, 2006). Sulfur 279 application increased the Cd concentrations in rice under low Cd level for DTPA-extractable) 280 while decreasing the Cd concentration in rice when sulfur was applied under higher Cd levels in 281 the soil (Gao et al., 2010). The increase in sulfur-mediated Cd uptake by plants might be due to 282 several reasons such as under anoxic environments sulfides are being formed, and the Cd is 283 sequestered along as Cd sulfide. When the sulfide is being oxidized to SO4, the Cd can be 284 liberated and thus can be transported to plants (Martinez et al., 2002; Karlsson et al., 2005; Gao 285 et al., 2010). 286 Plant available concentration of Cd in soil decreased with the addition of gypsum and MAP and 287 consequently, these treatments showed lowest Cd concentration in both soil depths (Table 3). 288 Few authors reported that gypsum and phosphate fertilizers (like MAP) show a higher affinity 289 for Cd fixation in the form of Cd phosphates, Cd carbonates, and various stable Cd complexes 290 and reduce plant-available Cd (Cui et al., 2016; Zhao et al., 2016). However, increase or 291 decrease in plant-available Cd concentrations in the soil varied with soil types, phosphate 292 fertilizers types and agricultural practices (Seshadri et al., 2016). In the present study, MAP 293 showed highly significant results for Cd immobilization for both depths (Figure 1C) which might 294 be due to higher availability of soluble phosphate that may form Cd phosphate and could 295 precipitate Cd in the soil. It has been reported that soluble sources of phosphorus, such as MAP 13 ACCEPTED MANUSCRIPT 296 might act as an agent for a higher Cd precipitation (Seshadri et al., 2016). Despite these benefits 297 of MAP, it can also be a source of Cd entry into the soil due to the presence of Cd in the rock 298 phosphate (Jiao et al., 2012; Niu et al., 2013; Murtaza et al., 2015). In such circumstances, 299 gypsum application might be a viable approach for Cd immobilization in the soil. It has been 300 observed that S application increased the bioavailable Cd concentration in soil compared to the 301 control (Table 3). Asgher et al. (2014) also reported that Cd and other metal concentrations 302 increased in the soil with sulfur application. 303 The residual MAP and gypsum increased the pH of the post-harvest soil compared to the control 304 (Table 3). There was also increase in pH of the same soil after harvesting of wheat and rice crops 305 as reported previously (Rehman et al., 2015). The present result confirms the persistent increase 306 of pH with MAP and gypsum. The increase or no change in soil pH with P application has been 307 reported in the literature which mainly depends upon soil types and cultivation practices (Jiang et 308 al., 2012; Seshadri et al., 2016). Furthermore, the increase in pH was negatively correlated with 309 soil AB-DTPA extractable soil Cd and Cd concentration in grains and straw of wheat (Figure 2). 310 This finding indicates that an increase in pH leads to a decrease in Cd concentration in the plant. 311 The increase in pH reduces the availability of Cd due to increased Cd precipitation and surface 312 adsorption on gypsum and MAP (Arshad et al., 2016). Furthermore, it has been reported that P 313 compounds could adsorb heavy metal(loid) such as Cd in the soil (Seshadri et al., 2016). In the 314 present study, S application decreased the soil pH compared to the control at both depths (Table 315 3). Previously, it has been reported that S has the potential to lower the pH (Vet et al., 2014). 316 Zhang et al. (2013) reported that S application provokes the S oxidation process and thus due to 317 acidic in nature, upon water availability release of H+ ions reduces soil pH. 14 ACCEPTED MANUSCRIPT 318 Cadmium uptake and transfer towards shoot and grains decreased by the application of gypsum 319 and MAP, whereas a contradicted response was observed in sulfur treatment (Table 2). This 320 effect is related to Cd immobilization factor that was a maximum for 0-15 cm with MAP @ 0.8 321 % (T4) and for 15-30 cm with MAP @ 0.4% (T3) while a minimum for 0-15 cm and for 15-30 322 cm with elemental sulfur @ 0.8 % (T10) (Figure 1). These results are in line with the finding of 323 Zeng et al. (2016) who reported reduced soil to shoot and shoot to grain Cd transfer with gypsum 324 and MAP application. More recently, it has been reported that flue gas desulphurization gypsum 325 has the potential to immobilize Cu and Pb in water and soil (Liu et al., 2016). Thus, a decrease in 326 Cd uptake and translocation with MAP and gypsum might be due to immobilization of Cd in the 327 soil and reduced its availability to wheat plant (Figure 1). Overall, the decrease in Cd 328 concentration in wheat plants indicates that these amendments, MAP and gypsum, has the long- 329 term ability to immobilize Cd in the soil. Residual S increased the Cd concentrations in wheat 330 straw and grains compared to the control (Table 2). The increase in Cd concentration in plants 331 with S application was also reported previously (Rehman et al., 2015). The increase in Cd 332 concentrations with S application might be due to the mobilization of the Cd in the soil (Dede 333 and Ozdemir, 2016). 334 The highest Cd harvest index was found with MAP followed by elemental S and gypsum 335 respectively (Figure 1B). This higher Cd harvest index with MAP and lower with gypsum might 336 be due to higher biomass production with MAP. This increase in biomass was concomitant with 337 a decrease in Cd concentration in plants which might be due to a dilution effect (same Cd 338 concentration but larger biomass) with MAP as suggested by Rizwan et al. (2012). However, in 339 the case of gypsum, the decrease in Cd concentration in plants (Table 2) could lead to less Cd 340 toxicity, and thus healthier plants and thus lower Cd harvest index was observed with this 15 ACCEPTED MANUSCRIPT 341 amendment. The higher Cd harvest index with S application might be due to higher Cd 342 concentrations in plants with S application (Table 2). The higher Cd harvest index indicated that 343 MAP and S can be used for phytoextraction purposes while the lower Cd harvest index with 344 gypsum indicated that this amendment might be used for phytostabilization of heavy metals in 345 the soil. 346 The benefit-cost ratio was highest in the control (Figure 3) and thus, its Cd contaminated grain 347 and straw is a risk to human health. Moreover, as no amendment was used in control, therefore 348 less expenditure (smaller denominator factor) increased the benefit-cost ratio. Among the 349 amendments, the maximum benefit-cost ratio was observed with gypsum @ 0.2% (T5). Although 350 the addition of MAP showed promising results concerning immobilization of Cd and amount of 351 yield, however, its lower benefit cost ratio coupled with higher prices may limit its use in 352 reducing Cd, probably other metals, uptake by plants (Marangoni et al., 216; Muhammed et al., 353 2016). 354 355 5. Conclusion 356 Gypsum and MAP had significant residual effects on decreasing Cd uptake by wheat plants 357 while S had residual effects on increasing Cd uptake by plants. Among the amendments, gypsum 358 increased the yield of wheat and reduced the Cd concentration in wheat grain, while MAP and 359 sulfur had low benefit-cost ratios. Thus, it can be recommended to use gypsum as an amendment 360 in Cd contaminated soils for growing food crops such as wheat which would be eco-friendly, 361 economical and proficient approach, especially in Cd-contaminated fields receiving raw city 362 effluent. 363 16 ACCEPTED MANUSCRIPT 364 Acknowledgments 365 Thanks to the University of Agriculture Faisalabad and Higher Education Commission of 366 Pakistan for financial support. 367 368 References 369 Adrees, M., Ali S., Rizwan, M., Ibrahim, M., Abbas, F., Farid, M., Rehman, M.Z., Irshad, M.K., 370 Bharwana, S.A., 2015a. The effect of excess copper on growth and physiology of 371 important food crops: a review. Environ. Sci. Pollut. Res. 22, 8148-8162. 372 Adrees, M., Ali, S., Rizwan, M., Rehman, M.Z., Ibrahim, M., Abbas, F., Farid, M., Qayyum, 373 M.F., Irshad, M.K., 2015b. Mechanisms of silicon-mediated alleviation of heavy metal 374 toxicity in plants: a review. Ecotoxicol. Environ. 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Pharmacol. 513 36, 1235-1241. 514 Zhao, T.T., Ge, W.Z., Yue, F., Wang, Y.X., Pedersen, C.M., Zeng, F.G., Qiao, Y., 2016. 515 Mechanism study of Cr (III) immobilization in the process of Cr (VI) removal by 516 Huolinhe lignite. Fuel Process. Technol. 152, 375-380. 23 ACCEPTED MANUSCRIPT Translocation index (%) 42 A ab 40 de cde abcd bcd abc a e 38 f g 36 34 32 30 0.45 B a Cd harvest index (%) 0.40 0.35 0.30 d b bc b e 0.25 f 0.20 cd b e 0.15 0.10 0.05 0.00 Cd immoblization (%) T1 1.0 T2 T4 T5 T6 T7 B C 0.6 0.4 T9 T10 D a 0-15 cm depth C a a D bc E c ab ab 0.0 -0.2 T8 A C 0.8 0.2 T3 T1 T2 T3 T4 T5 T6 T7 ab EF T8 T9 F d T10 d -0.4 -0.6 -0.8 Treatments G Figure 1. Shoot to grains Cd translocation index (A), Cd harvest index (B) and Cd immobilization in the soil (C) as affected by inorganic amendments containing mono-ammonium phosphate (MAP) @ 0.2 % (T2), @ 0.4 % (T3), @ 0.8 % (T4), Gypsum @ 0.2 % (T5), @ 0.4 % (T6), @ 0.8 % (T7) and sulfur @ 0.2 % (T8), @ 0.4 % (T9), @ 0.8 % (T10) and control (T1). Values are mean ± SE (n = 4). Different letters indicate significant difference between treatments at P ≤ 0.05 following Tukey-HSD. 1 0.45 Grain Cd (mg kg-1) AB-DTPA extractable Cd (mg kg-1) ACCEPTED MANUSCRIPT A 0.4 0.35 0.3 0.25 0.2 0.15 B 0.12 0.1 0.08 0.06 0.04 0.1 0.02 0.05 0 0 6.8 Straw Cd (mg kg-1) 0.14 0.3 7 7.2 7.4 Soil pH 7.6 7.8 8 7.4 7.6 Soil pH 7.8 8 6.8 7 7.2 7.4 Soil pH 7.6 7.8 8 C 0.25 0.2 0.15 0.1 0.05 0 6.8 7 7.2 Figure 2. Relationship between soil pH and soil AB-DTPA extractable Cd, Cd in grains and straw of wheat plants. 2 ACCEPTED MANUSCRIPT Benefit Cost Ratio 14 BCR 1st year BCR 2nd year BCR 3rd year 12 10 8 6 4 2 0 T1 T2 T3 T4 T5 T6 Treatments T7 T8 T9 T10 Figure 3. Cost benefit ratio of wheat grown in a Cd contaminated soil as affected by inorganic amendments containing mono-ammonium phosphate (MAP) @ 0.2 % (T2), @ 0.4 % (T3), @ 0.8 % (T4), Gypsum @ 0.2 % (T5), @ 0.4 % (T6), @ 0.8 % (T7) and sulfur @ 0.2 % (T8), @ 0.4 % (T9), @ 0.8 % (T10) and control (T1). 3 ACCEPTED MANUSCRIPT Table 1. Initial physicochemical properties of the soil without amendments under field experiment and the properties of the raw effluent used for irrigation. Physicochemical properties Soil Unit Parameter Sand % Silt % Clay % pHs ECe dS m-1 SAR (mmolc L-1)1/2 CEC cmolc kg-1 OM % Total Cd mg kg-1 Available Cd mg kg-1 * Ayers and Westcot (1985). Value 46 22 32 7.25 3.58 7.69 4.81 1.09 3.02 0.69 Parameter EC SAR Cd Zn Pb Co Cr Raw effluent Unit Value -1 dS m 1.5 (mmolc L-1)1/2 6.61 mg L-1 0.01 mg L-1 0.018 mg L-1 0.103 mg L-1 0.001 mg L-1 ND *Permissible limit < 1.5 < 7.5 ≤ 0.01 ≤ 2.0 ≤ 5.0 ≤ 0.05 ≤ 0.1 Table 2. Wheat straw and grain yield and Cd concentration and total Cd uptake by plants as affected by different inorganic amendments and irrigated with raw effluent. Values are mean ± SD (n = 4). Different letters indicate that values are significant different at P < 0.05. Treatments (T1) Control (T2) MAP @ 0.2 % (T3) MAP @ 0.4 % (T4) MAP @ 0.8 % (T5) Gypsum @ 0.2 % (T6) Gypsum @ 0.4 % (T7) Gypsum @ 0.8 % (T8) Sulfur @ 0.2 % (T9) Sulfur @ 0.4 % (T10) Sulfur @ 0.8 % Yield (Mg ha-1) Grain Straw e 3.39 ± 0.03 5.51 ± 0.08e cd 3.63 ± 0.06 5.81 ± 0.06d 3.83 ± 0.06b 5.99 ± 0.14bc b 3.98 ± 0.03 5.99 ± 0.13bc 3.8 ± 0.36bc 5.88 ± 0.08cd b 3.96 ± 0.06 6.08 ± 0.14ab 4.18 ± 0.03a 6.02 ± 0.05a e 3.41 ± 0.11 6.00 ± 0.07bc 3.55 ± 0.05de 5.87 ± 0.17cd 2.91 ± 0.08f 5.44 ± 0.14e Cd concentration (mg kg-1) Grain Straw b 0.10 ± 0.008 0.15 ± 0.009c d 0.08 ± 0.003 0.12 ± 0.004d 0.06 ± 0.006e 0.12 ± 0.006d e 0.05 ± 0.003 0.07 ± 0.004f 0.06 ± 0.001e 0.10 ± 0.005e e 0.05 ± 0.002 0.07 ± 0.006f 0.05 ± 0.004e 0.06 ± 0.004f c 0.09 ± 0.004 0.15 ± 0.006c 0.10 ± 0.004b 0.17 ± 0.002b a 0.13 ± 0.003 0.26 ± 0.009a Cd uptake (g ha-1) Grain Straw c 0.52 ± 0.03 0.84 ± 0.06c d 0.44 ± 0.02 0.70 ± 0.03d 0.47 ± 0.02d 0.73 ± 0.05d f 0.27 ± 0.01 0.41 ± 0.02f 0.38 ± 0.05e 0.58 ± 0.03e f 0.26 ± 0.02 0.40 ± 0.03f 0.27 ± 0.02f 0.40 ± 0.02f b 0.53 ± 0.04 0.93 ± 0.04b 0.59 ± 0.01b 0.97 ± 0.03b a 0.75 ± 0.03 1.40 ± 0.04a ACCEPTED MANUSCRIPT Table 3. Soil pHs and AB-DTPA extractable Cd in the soil as affected by different inorganic amendments and irrigated with raw effluent. Values are mean ± SD (n = 4). Different letters indicate that values are significant different at P < 0.05. Treatments (T1) Control (T2) MAP @ 0.2 % (T3) MAP @ 0.4 % (T4) MAP @ 0.8 % (T5) Gypsum @ 0.2 % (T6) Gypsum @ 0.4 % (T7) Gypsum @ 0.8 % (T8) Sulfur @ 0.2 % (T9) Sulfur @ 0.4 % (T10) Sulfur @ 0.8 % Soil pHs 0-15 cm 15-30 cm a 7.36 ± 0.01 7.39 ± 0.03a a 7.37 ± 0.09 7.41 ± 0.02a 7.39 ± 0.16ab 7.41 ± 0.03a ab 7.37 ± 0.22 7.41 ± 0.05a 7.36 ± 0.06a 7.49 ± 0.14a ab 7.44 ± 0.25 7.40 ± 0.02a 7.38 ± 0.01a 7.39 ± 0.04a b 7.29 ± 0.04 7.26 ± 0.13b bc 7.20 ± 0.14 7.19 ± 0.08bc 7.20 ± 0.07c 7.20 ± 0.05c AB-DTPA extractable Cd (mg kg-1) 0-15 cm 15-30 cm c 0.26 ± 0.011 0.26 ± 0.004b d 0.19 ± 0.004 0.21 ± 0.007d 0.12 ± 0.004e 0.20 ± 0.011d g 0.02 ± 0.002 0.21 ± 0.007d 0.19 ± 0.006d 0.25 ± 0.037bc e 0.12 ± 0.006 0.23 ± 0.034cd 0.08 ± 0.008f 0.23 ± 0.019cd c 0.26 ± 0.017 0.22 ± 0.012c b 0.28 ± 0.012 0.29 ± 0.006b 0.42 ± 0.008a 0.30 ± 0.005a