International Journal of Phytoremediation, 17: 165–174, 2015 C Taylor & Francis Group, LLC Copyright  ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2013.876962

Evaluation of Organic and Inorganic Amendments on Maize Growth and Uptake of Cd and Zn from Contaminated Paddy Soils NARUPOT PUTWATTANA1, MALEEYA KRUATRACHUE1,2, ACHARAPORN KUMSOPA3, and PRAYAD POKETHITIYOOK1 1

Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand Mahidol University International College, Mahidol University, Nakhon Pathom, Thailand 3 Faculty of Environment and Resource Studies, Mahidol University, Nakhon Pathom, Thailand 2

Pot and field experiments were conducted to investigate the effects of soil amendments (cow manure, rice straw, zeolite, dicalcium phosphate) on the growth and metal uptake (Cd, Zn) of maize (Zea mays) grown in Cd/Zn contaminated soil. The addition of cow manure and rice straw significantly increased the dry biomass, shoot and root length, and grain yield of maize when compared with the control. In pot study, cow manure, rice straw, and dicalcium phosphate all proved effective in reducing Cd and Zn concentrations in shoots and roots. Cd and Zn concentrations in the grains of maize grown in field study plots with cow manure and dicalcium phosphate amendments to highly contaminated soil (Cd 36.5 mg kg−1 and Zn 1520.8 mg kg−1) conformed to acceptable standards for animal feed. Additionally both cow manure and dicalcium phosphate amendments resulted in the significant decrease of Cd and Zn concentrations in shoots of maize. Keywords: Cd, Zn, amendments, maize (Zea mays), pot experiment, field experiment

Introduction Heavy metal contamination of agricultural soils is a major environmental concern due to its detrimental effects on plant productivity and soil ecosystems, and because of health risk posed by the contamination of crops grown either as food or as animal feed (Zheng et al. 2007; Zheljazcov et al. 2008). Cadmium (Cd) and zinc (Zn) are frequent heavy metal contaminants of agricultural soils (Alloway 1990). For agronomic crops, the approximate concentrations of Cd and Zn in mature leaf tissue which are excessive or toxic were reported as 5–30 mg kg−1 and 100–400 mg kg−1, respectively (KabataPendias 2001). In one such heavy metal (Cd and Zn) contaminated area is in Mae Sod district, Tak province, western Thailand, paddy fields receiving irrigation from the two creeks (Mae Tao and Mae Ku) were found to contain markedly elevated Cd levels during surveys implemented in 2001–2004 (Simmons et al. 2005). Total soil Cd and Zn concentrations were 0.5–284 mg kg−1 and 100-8036 mg kg−1, respectively (Simmons et al. 2005; Phaenark et al. 2009). While the soils remained fertile, farming in these areas produced rice grains and soybeans with elevated

Address correspondence to M. Kruatrachue, Department of Biology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. E-mail: [email protected]

Cd contents (> 0.2 mg kg−1 standard according to the Codex Committee on Food Additives and Contaminants, CCFAC), which were unfit for human or animal consumption (Simmons et al. 2005). Elevated levels of urinary Cd were found in Cdexposed populations in Mae Sod (Swaddiwudhipong et al. 2007; Teeyakasem et al. 2007) and rice cultivation has been prohibited in an effort to prevent further exposure. Even so, the government has found it difficult to regulate local agricultural practices, and many farmers continue to grow high risk crops. Feasible alternatives might be either to substitute crops which are not consumed by humans, or reduce metal concentrations in plants by immobilizing heavy metals in soils through the addition of organic or inorganic amendments. Soil amendments may be crucial in both reducing Cd uptake by plants grown in Cd contaminated soils, and increasing plant biomass. Silicon (Si) sources such as zeolites have great potential for Cd immobilization in plants and were reported to significantly reduce the Cd uptake and accumulation in plants (Chlopecka and Adriano 1997; Keller et al. 2005; Hamidpour et al. 2010). Si significantly reduced Cd uptake and accumulation in plants in both hydroponic and pot studies (Shi et al. 2005; Liang et al. 2005; Putwattana et al. 2010). Hodson et al. (2000) reviewed the evidence showing the formation of metal phosphates in phosphate-treated metal-contaminated soils. Addition of hydroxyapatite markedly reduced Cd concentration in the leaves of tobacco (Keller et al. 2005) and African basil (Chaiyarat et al. 2011). Organic amendments

166 such as animal manure can reduce Cd uptake and accumulation in crop plants in both pot and field experiments (Li et al. 2006; Sato et al. 2010; Chaiyarat et al. 2011). The ability to absorb heavy metals differs between crop species. It is important to select crop plants with a low ability to absorb heavy metals. In particular, metal concentrations in the edible parts of plant have to be below the maximum limits of heavy metals in foods. In the Mae Sod area, maize has been grown after alternate rice-harvesting seasons. Several studies attempted to decrease the concentration of heavy metals especially Cd in the maize grain using various soil amendments such as zeolite, apatite, lime, municipal solid waste (Chlopecka and Adriano 1997; Guo et al. 2011; Carbonell et al. 2011). While substantial amounts of Cd were observed in the roots, Cd levels in grains were negligible. The aims of this study were to investigate the effects of various organic (cow manure and rice straw) and inorganic amendments (zeolite and dicalcium phosphate) on Cd and Zn uptake and accumulation in maize grown in pot and field experiments, and to determine the suitability of maize as alternative crop for metal contaminated soil.

N. Putwattana et al. Table 1. Soil treatment used on Cd contaminated soil in pot experiment Addition amount in % (w/w)

Abbreviation

Control soil Cd soil

— —

Cont Cd

Cd soil with cow manure 1

10

CdCw10%

Cd soil with cow manure 2

20

CdCw20%

Cd soil with dicalcium phosphate 1

1

CdDi1%

Cd soil with dicalcium phosphate 2

5

CdDi5%

Cd soil with zeolite 1

1

CdZeo1%

Cd soil with zeolite 2

5

CdZeo5%

Treatment

Cd soil with rice straw 1

6.25

CdSt 6.25%

Cd soil with rice straw 2

12.5

CdSt 12.5%

Materials and Methods Pot Experiment Soil samples used in treatment groups in the pot experiments were taken from the surface layer (10–20 cm depth) of a Cdcontaminated paddy field in Phadae village, Mae Sod district, Tak province, Thailand (N 16◦ 40’ 35.9˝a E 98◦ 37’ 37.4˝a). Agricultural soil purchased from a local store in the village was used for the control group. Cd-contaminated soils were treated with organic soil additives (cow manure and rice straw) and inorganic additives [dicalcium phosphate (CaHPO4 ) and zeolite ((Na,K,Ca)4 Al6 Si30 O72 .24H2 O)]. Cow manure and rice straw were obtained from pasture land in Mae Sod district. Cow manure was air dried and sieved through a 2-mm mesh whereas rice straw was chopped into 2-cm pieces. Dicalcium phosphate was the form used for feeding livestock, and purchased from the JFK feed shop in Nakhon Pathom province. Zeolite was obtained from the Otopkano distributor located in Nakhon Ratchasima province. The experiment was performed with 10 treatments and 4 replicates per treatment (Table 1). A basal chemical fertilizer (N:P:K = 16:8:8) mixed thoroughly with the 2 kg air dried soil (1g kg−1) was applied to each treatment. The soil samples (2 kg) were placed in each plastic pot (10 cm in diameter and 20 cm in height). The soil additives were mixed with the soil samples thoroughly and equilibrated with deionized water one month prior to planting. Maize (Zea mays) were obtained from the Crop Research Center, Tak Fa district, Nakhon Sawan province. Five seeds were sown directly into each pot. The pots were kept in the greenhouse under controlled conditions (25–28◦ C, natural sunlight, 12/12 h photoperiod, 60% relative humidity) on benches in a randomized complete block design. Seven days after sowing, each pot was thinned to two seedlings. Plants were watered by means of sprinklers every other day. Growth

continued for 3 months. Plant and soil samples were collected after 3 months.

Field Experiment The study was conducted in Cd-contaminated paddy field in Phadae village, Mae Sod district during January–March 2012. The climate is tropical, with a mean annual temperature of 26.5◦ C and an average annual rainfall of 2052 mm. Four different sites each containing different concentrations of Cd and Zn were selected : site A, high (Cd 36.5 mg kg−1, Zn 1520.8 mg kg−1); site B, moderate (Cd 10.4 mg kg−1, Zn 697.9 mg kg−1); site C, low (Cd 4.7 mg kg−1, Zn 415.7 mg kg−1); and site D, control (Cd 1.5 mg kg−1, Zn 128.8 mg kg−1). Four plots of 2 × 2 m each were set up at each site. They represented 4 treatments: (1) control, consisting of Cd contaminated soil only; (2) 20% w/w cow manure; (3) 12.5% w/w rice straw; (4) 5% w/w dicalcium phosphate. Three replications were performed in each treatment. Each plot was split into 4 rows; each row consisting of 3 plants. Each plot treatment contained 12 plants; 48 plants were used in total for each site. Cow manure was added at 390 Megagram hectare−1 (Mg ha−1) while rice straw was added at 487.5 Mg ha−1. Dicalcium phosphate was provided at a dose of 97.5 Mg ha−1. The soil in each plot was ploughed to a 20 cm depth and tilled. After the addition of basal chemical fertilizer at a dose of 200 kg ha−1, seeds were sown at density of 15 seeds (m2)−1. The plots were watered by a field irrigation channel which allowed water to flow in every two days. Urea fertilizer was applied at the rate of 125 kg ha−1 after 1 month. Weeds were removed manually during the crop cycle. Plants were harvested using randomized complete block design (RCBD) after 3 months of growth. Soil samples (depth, 0–20 cm) were collected from

Maize Growth and Uptake of Cd and Zn from Paddy Soils each plot after 1 and 3 months of treatment. They were mixed to obtain a composite sample before analysis.

167 software. Least Significant Difference (LSD) was used to detect the significant difference among means of different treatments.

Chemical Analysis Soils (agricultural and Cd-contaminated soils), cow manure, rice straw, dicalcium phosphate, and zeolite were characterized using standard procedures. pH was determined by a pH meter (HI 221, Hanna Instruments), organic matter by Walkley-Black titration (Walkley and Black 1934), cation exchange capacity (CEC) by sodium saturation (Chapman 1965), total N by the Kjeldhal method (Black 1965), available P by the Bray II method (Bray and Kurtz 1945), available K by an atomic absorption spectrophotometer after extraction with NH4 OAc (ICARDA 2001), Ca and Mg by the atomic absorption spectrophotometer with 1 N ammonium acetate, pH 7.0 (Pratt 1965). Soil and additives were air-dried, ground and passed through 2-mm nylon mesh sieve. They were digested in aqua regia according to Ure (1995). After harvest, the plants were measured for shoot and root length. They were washed with tap water followed by deionized water, divided into shoots (leaves and stem), root, flower, grain and cob. These were dried at 85◦ C in the oven for 5 days and their dry weights recorded. Plant samples were ground with mortar into fine powder and sieved through a 2-mm nylon mesh sieve. Dry plants were digested with the mixture of nitric/ perchloric acid (2:1 v/v) (Johnson and Ulrich 1959). After digestion, both plant and soil samples were filtered through a Whatman No. 42 filter paper. Cd and Zn concentrations were analyzed by a flame atomic absorption spectrophotometer (FAAS, Variance Spectra AA 55 B). Cd and Zn availability in the soil were determined using diethylenetriamine-penta-acetic acid (DTPA) extraction (Simmons et al. 2005).

Data Analysis The translocation factor (TF), the plant’s ability to translocate heavy metals from root to shoot or the harvestable aerial parts (Mattina et al. 2003), was calculated as follows: TF =

Concentration of metal in aerial parts (mg kg−1 ) Concentration of metal in roots (mg kg−1 )

The bioconcentration factor for roots (BCFR), which provides an index of the ability of the plant roots to accumulate the metal with respect to the metal concentration in the substrate (Mane et al. 2010), was calculated as follows: BCFR =

Concentration of metal in roots (mg kg−1 ) Concentration of metal in soil (mg kg−1 )

Statistical Analysis Data were analyzed using analysis of variance (ANOVA) at significance level of P≤ 0.05 with SPSS 18.0 computer

Results Chemical Properties of Soils and Amendments The pH of all soils was relatively neutral except for Cd soil in the pot study (pH 7.8, Table 2). In comparison, the agricultural soil had high organic matter (OM), cation exchange capacity (CEC), total Ca, total Mg and total K than the other soils. Soil at site D (control plot in the field study) contained the lowest OM, CEC, total Ca, total Mg and total K. Cd soil contained the highest Cd (50.7 mg kg−1) and Zn concentrations (1984.8 mg kg−1) (Table 2). The amendments showed different chemical properties. Dicalcium phosphate was acidic (pH 4.2) while the pH of zeolite and cow manure was alkaline. The richest organic matter was found in rice straw followed by cow manure. Zeolite had the highest CEC, but the lowest total N, P, and K. Dicalcium phosphate had the highest concentrations of total P and total Ca while total K was not detected. Among the soil additives, cow manure contained the highest total N, total Mg, and total K (Table 2). Effects of Amendments on Bioavailability of Cd and Zn In the pot experiment, the application of 5% dicalcium phosphate significantly reduced extractable Cd by 1.8-fold compared with Cd/Zn soil (P ≤ 0.05). The addition of 5% zeolite resulted in the 1.2-fold decrease of extractable Zn (P ≤ 0.05, Table 3). The field experiment showed slightly different results (Table 4). The application of dicalcium phosphate, cow manure, and rice straw significantly decreased extractable Cd in soil at site A with the highest concentrations of Cd and Zn, site C (low Cd and Zn concentrations), and control site (P ≤ 0.05). However, significant decrease in extractable Zn was observed at site A (rice straw and cow manure treatments) and control site (all treatments). Effects of Amendments on the Growth of Maize In the pot study, amendments other than zeolite increased the dry biomass, shoot and root length of plants compared to those grown on Cd/Zn contaminated soil alone (P ≤ 0.05, Table 5). The order of growth increase was rice straw > cow manure ∼ dicalcium phosphate. The increases in dry biomass, shoot length and root length were 4.5-fold, 1.4-fold, and 2.8fold, respectively in the 12.5% rice straw treatment. The field trial study revealed different results (Table 6). Growth in total dry biomass was greater (P ≤ 0.05) at site C (low Cd/Zn concentrations) and site D (control site) with the addition of rice straw and cow manure, while root length was similar in all four treatments (P > 0.05). At site A (highest Cd/Zn concentrations), plants showed no increase in growth (P > 0.05). The greatest growth in dry biomass, shoot length

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Table 2. Chemical properties of soils and amendments used in pot and field experiment Parameter

Agricultural soil

Cd soil

A

B

C

D

Di

Zeo

Cw

St

7.3 3 38.4 0.13 0.11 25 5854 654 122 0 273.5

7.8 2.4 13.2 0.11 0.09 6 4952 251 69 50.7 1984.8

6.9 2.9 15.2 1.69 0.15 17 5512 388 0.57 36.5 1520.8

7.4 1.83 12.2 1.17 0.09 19 2565 284 0.32 12.5 697.9

7.2 1.8 12 1.2 0.1 160 1992 243 0.15 4.7 415.7

6.9 1.5 9.2 1.85 0.08 24 1391 197 0.12 1.5 128.8

4.2 — 8 2.72 0.21 218300 200400 3300 nd 1.3 109.7

8.3 — 137.4 0.1 0.09 nd 35 1600 4100 0.5 14.2

8.3 27.58 42 3.58 1.75 12400 14000 9800 45500 1.2 103.3

6.8 68.84 — 4.26 1.05 22000 4400 2000 18800 0.6 12.6

pH Organic matter(%) CEC (cmol kg−1) EC (ds m−1) Total N (%) Total P (mg kg−1) Total Ca (mg kg−1) Total Mg (mg kg−1) Total K (mg kg−1) Total Cd (mg kg−1) Total Zn (mg kg−1)

A = high Cd contaminated site, B = moderate Cd contaminated site, C = low Cd contaminated site, D = control site, Di = dicalcium phosphate, Zeo = zeolite, Cw = cow manure, St = rice straw.

Table 3. Total concentration and DTPA-extractable Cd and Zn in soils with various additives in pot experiment after 3 months Soil treatment

Total Cd

Extractable Cd

Control Cd Cd cw 10% Cd cw 20% Cd Di 1% Cd Di 5% Cd Zeo 1% Cd Zeo 5% Cd Str 6.25% Cd Str 12.5%

0.0 ± 0.0 48.5 ± 1.6a 44.4 ± 1.1be 42.3 ± 0.9b 46.8 ± 1.8ac 43.2 ± 0.9b 46.2 ± 2.6cde 45.8 ± 1.8cd 48.2 ± 0.5ae 47.1 ± 1.8ac

0.0 ± 0.0 16.5 ± 2.0ae 15.1 ± 3.9ace 13.5 ± 2.8ace 13.1 ± 1.3bc 9.1 ± 1.6bc 17.7 ± 1.9e 14.0 ± 5.1ade 13.2 ± 3.2ab 13.1 ± 2.1ade

Total Zn

Extractable Zn

307.2 ± 51.4a 2032.8 ± 56.8bd 1850.3 ± 139.7bc 1804.8 ± 144.5c 1915.9 ± 114.2bc 1884.4 ± 331.6bc 2012.8 ± 124.9bcd 1966.6 ± 80.4bcd 2146.4 ± 151.6d 1912.7 ± 149.0bc

2.5 ± 0.5a 199.5 ± 13.1bd 191.5 ± 4.0bd 195.0 ± 3.2bd 205.5 ± 10.0b 188.0 ± 19.8bde 170.9 ± 12.1ce 165.4 ± 22.4c 198.3 ± 17.1bd 184.5 ± 9.6de

Values followed by the same letter did not differ; small letters showed differences between amendment effects (P ≤ 0.05, LSD).

Table 4. Total concentration and DTPA-extractable Cd and Zn in soils with various additives in field trial experiment after 3 months Site A

B

C

D

Soil treatment

Total Cd

Extractable Cd

Total Zn

Extractable Zn

Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20%

41.2 ± 0.6a 37.2 ± 3.5b 33.2 ± 2.9c 26.8 ± 2.1d 8.5 ± 3.5a 10.7 ± 1.3a 11.0 ± 0.7a 9.8 ± 0.5a 4.0 ± 0.5a 3.4 ± 0.2a 3.5 ± 0.2a 3.4 ± 0.5a 1.5 ± 0.3a 1.5 ± 0.3a 1.1 ± 0.3ab 1.0 ± 0.1b,B

16.8 ± 1.9a 12.8 ± 0.9b 15.2 ± 1.9ac 13.6 ± 0.5bc 5.8 ± 0.2a 5.9 ± 0.5ab 6.3 ± 0.1b 6.0 ± 0.1ab 4.1 ± 0.2a 2.9 ± 0.3b 3.3 ± 0.2c 3.1 ± 0.3bc 2.0 ± 0.2a 1.5 ± 0.2b 1.5 ± 0.2b 1.2 ± 0.1b

1340.0 ± 80.6a 1107.0 ± 86.3b 1095.5 ± 84.3b 971.9 ± 131.6b 380.6 ± 83.8a 434.9 ± 74.3a 433.1 ± 48.6a 440.6 ± 16.4a 243.7 ± 48.8a 244.6 ± 16.1a 239.7 ± 44.7a 264.6 ± 41.4a 154.1 ± 51.7a 162.6 ± 25.2a 150.9 ± 19.0a 125.5 ± 65.0a

142.3 ± 12.1ab 152.9 ± 15.7a 120.9 ± 21.5b 125.7 ± 11.0b 55.8 ± 4.7a 68.5 ± 7.1b 60.8 ± 2.9ab 54.4 ± 10.8a 53.6 ± 9.3a 45.6 ± 13.2a 53.3 ± 5.1a 51.1 ± 2.3a 38.2 ± 5.8a 25.5 ± 1.5b 26.6 ± 1.5b 21.3 ± 3.1b

Values followed by the same letter did not differ; small letters showed differences between amendment effects within the same sites (P ≤ 0.05, LSD).

Maize Growth and Uptake of Cd and Zn from Paddy Soils Table 5. Dry biomass, shoot length and root length of maize grown with various soil additives in pot experiment after 3 months Soil treatment Control Cd Cd cw 10% Cd cw 20% Cd Di 1% Cd Di 5% Cd Zeo 1% Cd Zeo 5% Cd Str 6.25% Cd Str 12.5%

Dry mass (g)

Shoot length (cm)

Root length (cm)

2.0 ± 0.7a 4.1 ± 2.0a 14.5 ± 2.2bc 16.7 ± 3.9bc 12.9 ± 4.4c 14.9 ± 3.4bc 5.1 ± 4.1a 4.7 ± 2.7a 13.9 ± 5.7bc 18.6 ± 3.0b

53.1 ± 7.2a 84.5 ± 17.7bd 105.7 ± 3.8cf 118.0 ± 9.8c 96.5 ± 15.8df 116.5 ± 9.7c 61.3 ± 16.3ae 79.3 ± 15.1de 97.0 ± 19.3df 116.5 ± 9.8c

35.3 ± 17.2abd 20.0 ± 5.9ad 32.3 ± 4.7ab 43.0 ± 4.8bc 38.3 ± 7.0ab 28.8 ± 9.0abd 28.3 ± 10.8ad 16.8 ± 3.9d 55.5 ± 9c 39.8 ± 4.6b

Values followed by the same letter did not differ; small letters showed differences between amendment effects (P ≤ 0.05, LSD).

and ear dry mass was observed in plants grown at the control site with the addition of cow manure (dry biomass 305.4 g plant−1, shoot length 222.5 cm, ear dry mass 153.1g; Table 6). However, while the increase in dry biomass in the field study was 2.5-fold lower than that in the pot study, the increase in shoot length was similar in both (Tables 5 and 6). The cow manure treatment gave the greatest increase in ear dry biomass and grain yield followed by the rice straw treatment (a 2.4-fold increase at sites C and D, a 1.1–1.4-fold at sites A and B; Table 6).

Effects of Amendments on the Accumulation of Cd and Zn In the pot study, when all the treatments were compared with the Cd/Zn treatment, cow manure, dicalcium phosphate,

169 and rice straw addition resulted in significantly lower root (15–18 mg kg−1) and shoot (2–2.5 mg kg−1) accumulation. Similar results were also observed with Cd accumulation in the ear (1.2–1.7 mg kg−1). The field results demonstrated that both metal concentrations in plants increased with increased concentrations in the soil (Tables 8 and 9). Cow manure and dicalcium phosphate amendments yielded the lowest Cd concentration in plants at site A (P ≤ 0.05, Table 8). At sites B and C, cow manure resulted in the lowest shoot accumulation. Cd accumulation in both cob and grain were in the range of 0.1–0.2 mg kg−1 in maize grown on high and moderate Cd/Zn contaminated soils (sites A and B). At the control site, Cd was not detected in either cob or grain. The pot study revealed that cow manure and rice straw amendments caused the lowest Zn accumulation in both roots and shoots (Table 7). In the field study, cow manure and dicalcium phosphate were very effective in reducing Zn accumulation in shoots and roots of plants grown in soil at site A (Table 9). However, at sites B, C, and D, dicalcium phosphate and rice straw amendments were more effective. Cow manure addition resulted in increased Zn accumulations in all plant parts, probably due to the increased biomass. Zn accumulation in cob and grain was in the range of 13.2–56.0 mg kg−1 and 24.1–40.8 mg kg−1, respectively (Table 9). The lowest Zn accumulation in cob was 13.2 mg kg−1 resulting from rice straw amendment, and in seeds 24.1 mg kg−1 as a result of dicalcium phosphate amendment (Table 9). In both pot and field trial experiments, the roots always showed higher Cd concentrations than the shoots (translocation factor, TF1, indicating the translocation of Zn from roots to shoots (Table 9).

Table 6. Dry biomass, shoot length and root length of maize grown with various soil additives in field trial experiment after 3 months Site A

B

C

D

Soil treatment

Dry mass (g)

Ear dry mass (g)

Shoot length (cm)

Root length (cm)

Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20%

100.7 ± 30.5a,A 124.4 ± 38.9a,A 154.8 ± 19.3a,A 136.2 ± 53.0a,A 177.8 ± 86.2a,B 165.7 ± 22.5a,A 224.0 ± 51.8a,B 237.5 ± 54.6a,BC 103.6 ± 33.1a,AB 122.3 ± 21.9a,A 205.0 ± 54.8b,AB 221.3 ± 54.2b,B 122.7 ± 20.1a,AB 137.5 ± 72.9ab,A 198.7 ± 34.0b,AB 305.4 ± 48.8c,C

52.2 ± 17.0a,A 70.7 ± 26.8ab,A 96.2 ± 13.0b,A 75.3 ± 34.5ab,A 121.3 ± 7.6ab,B 100.2 ± 15.5a,A 118.5 ± 26.0ab,A 131.6 ± 22.4b,B 51.7 ± 31.2a,A 69.6 ± 15.3a,A 111.8 ± 16.9b,A 123.5 ± 32.6b,ABC 64.8 ± 16.0a,A 73.7 ± 45.6a,A 97.4 ± 24.3a,A 153.1 ± 40.2b,C

158.3 ± 12.4a,ABC 165.8 ± 18.4a,ABC 178.0 ± 11.5a,A 168.8 ± 11.4a,A 181.0 ± 15.4a,AB 189.8 ± 8.7ab,AB 223.5 ± 16.4c,B 206.0 ± 12.7bc,B 141.8 ± 21.4a,C 191.0 ± 23.5b,B 210.3 ± 17.9b,B 210.5 ± 13.0b,B 171.5 ± 6.1a,A 147.0 ± 20.6b,C 208.0 ± 16.2c,B 222.5 ± 6.6c,B

18.0 ± 4.0a,A 21.0 ± 3.7a,A 21.5 ± 1.9a,A 20.3 ± 1.3a,A 20.0 ± 3.6a,AB 20.5 ± 2.6a,A 24.0 ± 1.8a,AB 22.0 ± 2.2a,A 29.0 ± 4.2a,C 22.0 ± 2.2b,A 23.5 ± 4.7ab,AB 25.0 ± 4.9ab,A 24.0 ± 3.2a,BC 26.5 ± 11.0a,A 26.8 ± 2.5a,B 28.5 ± 10.3a,A

Values followed by the same letter did not differ; small letters showed differences between amendment effects within the same site (P ≤ 0.05, LSD), capital letters demonstrated the difference between sites of plant grown in the same treatment (P ≤ 0.05, LSD). Data was represented as means ± standard deviation (n = 4).

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Table 7. Cd and Zn accumulation, translocation factor (TF) and bioconcentration factor for roots (BCFR) of maize grown with various soil additives in pot experiment after 3 months Shoot

Root

Ear

TF

BCFR

Soil treatment

Cd

Zn

Cd

Zn

Cd

Zn

Cd

Zn

Cd

Zn

Control Cd Cd cw 10% Cd cw 20% Cd Di 1% Cd Di 5% Cd Zeo 1% Cd Zeo 5% Cd Str 6.25% Cd Str 12.5%

0.0 ± 0.0 6.0 ± 2.0a 2.5 ± 1.0b 2.0 ± 0.4b 4.2 ± 0.7a 2.4 ± 0.2b 8.0 ± 1.7a 6.8 ± 3.6a 4.0 ± 1.1a 2.5 ± 0.3b

72.8 ± 14.5a 168.8 ± 35.4b 85.8 ± 43.2a 62.2 ± 8.9a 164.9 ± 31.9b 183.1 ± 54.5b 84.9 ± 13.4ac 66.6 ± 7.9ac 56.9 ± 21.1a 73.0 ± 26.4ac

0.0 ± 0.0 30.2 ± 4.8ac 15.6 ± 4.0b 15.7 ± 3.6b 22.6 ± 7.5ac 18.1 ± 6.2b 39.1 ± 13.6c 31.2 ± 7.4ac 24.0 ± 5.5ab 28.5 ± 14.0ac

62.0 ± 5.7a 501.6 ± 108.6b 280.8 ± 85.4cd 234.7 ± 80.3d 358.4 ± 102.0bcd 304.6 ± 97.9cd 404.4 ± 34.7bcd 426.1 ± 151.5bc 329.9 ± 71.2cd 240.7 ± 119.9d

— 2.9 ± 0.9a 1.2 ± 0.6b 1.3 ± 0.7b 1.4 ± 0.5b 1.2 ± 0.2b — — 1.5 ± 0.2b 1.7 ± 0.3b

— 74.0 ± 14.7a 56.6 ± 7.8a 46.2 ± 18.0b 47.8 ± 17.2b 45.4 ± 6.1b — — 52.7 ± 20.0a 64.0 ± 13.0a

— 0.30 0.24 0.22 0.26 0.22 0.22 0.24 0.25 0.17

1.05 0.49 0.54 0.51 0.63 0.84 0.19 0.17 0.34 0.64

— 0.62 0.35 0.37 0.48 0.42 0.85 0.68 0.50 0.60

0.20 0.25 0.15 0.13 0.19 0.16 0.20 0.22 0.15 0.13

Values followed by the same letter did not differ; small letters showed differences between amendment effects (P ≤ 0.05, LSD).

In the pot study, higher BCFR values of Cd and Zn accumulation were observed in the Cd/Zn and zeolite treatments (Table 7). The BCFR values of root accumulation were always higher than those of the shoot and ear. Similar trends were observed in plants grown in the field (Tables 8 and 9).

Discussion Chemical Properties of Soils and Amendments The heterogeneity of Cd and Zn in the Mae Sod ricebased agricultural area was demonstrated by Simmons et al. (2005) which further indicated that Cd/Zn contamination was associated with suspended sediment transported to paddy

fields via the irrigation supply. Consequently, the spatial distribution of Cd and Zn was directly related to a field’s proximity to the irrigation canal containing suspended sediment. Phaenark et al. (2009) reported extremely high concentrations of Cd (542.6–894 mg kg−1) and Zn (20,271–31319 mg kg−1) in soils of Padaeng Zn mine located near the Padae village. Fuge et al. (1993) determined up to 980 mg kg−1 Cd in soils near metalliferous mines, and Peters and Shem (1992) measured 900–1,500 mg kg−1 Cd in soils near smelting operations. Other extremely high Zn concentrations were also reported, for example, Zwonitzer et al. (2003) reported 29,100 mg kg−1 Zn in an urban soil, and Skokart et al. (1983) found up to 180,000 mg kg−1 Zn in soil from a metal processing industry.

Table 8. Cd accumulation, translocation factor (TF) and bioconcentration factor for roots (BCFR) of maize grown with various soil additives in field trial experiment after 3 months Site A

B

C

D

Soil treatment

Shoot

Root

Flower

Cob

Grain

Ear

TF

BCFR

Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20%

4.6 ± 0.2a,A 2.8 ± 0.2b,A 4.4 ± 1.0a,A 2.9 ± 0.3b,A 2.1 ± 0.4a,B 2.1 ± 0.1a,B 1.8 ± 0.3ab,B 1.5 ± 0.3b,B 3.3 ± 0.6a,C 1.8 ± 0.3bc,C 1.9 ± 0.3b,B 1.3 ± 0.1c,B 0.7 ± 0.1a,D 1.1 ± 0.1b,D 0.0 ± 0.0c,C 0.0 ± 0.0c,C

21.4 ± 3.0a,A 11.2 ± 0.2b,A 15.0 ± 4.0b,A 13.8 ± 6.2b,A 9.9 ± 4.0a,B 6.3 ± 1.6a,B 9.6 ± 1.9a,B 9.2 ± 1.1a,A 2.2 ± 0.5a,C 1.7 ± 0.7a,B 2.7 ± 0.6a,BC 2.0 ± 0.6a,BC 1.7 ± 1.0a,C 1.7 ± 0.3a,C 1.2 ± 0.4b,C 2.1 ± 0.7a,C

1.8 ± 0.6ab,A 1.6 ± 0.2a,A 2.3 ± 0.6b,A 1.8 ± 0.3ab,A 1.7 ± 0.0a,A 1.7 ± 0.1ab,A 2.3 ± 0.5b,A 1.6 ± 0.2b,A 0.7 ± 0.5a,B 0.4 ± 0.3a,B 0.4 ± 0.2a,B 0.4 ± 0.3a,B 0.5 ± 0.3a,B 0.7 ± 0.4b,C 0.1 ± 0.1c,B 0.0 ± 0.0c,C

0.1 ± 0.1a,A 0.1 ± 0.0a,A 0.1 ± 0.0a,A 0.1 ± 0.0a,A 0.1 ± 0.0ab,A 0.1 ± 0.0a,A 0.2 ± 0.0b,B 0.1 ± 0.0a,A 0.4 ± 0.0a,B 0.2 ± 0.0b,B 0.3 ± 0.1ab,C 0.2 ± 0.1b,B 0.0 ± 0a,C 0.0 ± 0a,C 0.0 ± 0a,D 0.0 ± 0a,C

0.1 ± 0.0a,A 0.0 ± 0.0b,A 0.1 ± 0.0a,A 0.0 ± 0.0b,A 0.0 ± 0.0a,B 0.0 ± 0.0a,A 0.0 ± 0.0a,B 0.0 ± 0.0a,A 0.2 ± 0.1ab,C 0.2 ± 0.0bc,B 0.1 ± 0.0c,C 0.2 ± 0.0a,B 0.0 ± 0a,B 0.0 ± 0a,A 0.0 ± 0a,B 0.0 ± 0a,A

0.8 ± 0.0a,A 0.8 ± 0.1a,A 0.8 ± 0.1a,A 0.9 ± 0.0a,A 0.6 ± 0.1a,B 0.7 ± 0.1a,A 0.7 ± 0.0a,A 0.6 ± 0.0a,B 1.2 ± 0.1a,C 0.4 ± 0.1b,B 0.4 ± 0.1b,A 0.5 ± 0.1b,A 0.0 ± 0.0a,D 0.0 ± 0.0a,C 0.0 ± 0.0a,A 0.0 ± 0.0a,B

0.22 0.25 0.30 0.21 0.21 0.34 0.19 0.17 0.58 0.36 0.30 0.21 0.27 0.61 0.00 0.00

0.52 0.30 0.45 0.52 1.16 0.59 0.87 0.94 1.42 1.47 1.89 1.77 1.84 1.26 3.42 2.70

Values followed by the same letter did not differ; small letters showed differences between amendment effects within the same site (P ≤ 0.05, LSD), capital letters demonstrated the difference between sites of plant grown in the same treatment (P ≤ 0.05, LSD). Data was represented as means ± standard deviation (n = 4).

Maize Growth and Uptake of Cd and Zn from Paddy Soils

171

Table 9. Zn accumulation, translocation factor (TF) and bioconcentration factor for roots (BCFR) of maize grown with various soil additives in field trial experiment after 3 months Soil Site treatment A

B

C

D

Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20% Cd CdDi5% CdSt12.5% CdCw20%

Shoot

Root

Flower

Cob

Grain

Ear

294.1 ± 45.2a,A 216.5 ± 15.6b,A 254.7 ± 53.2ab,A 225.7 ± 16.2b,A 185.8 ± 24.9a,B 123.3 ± 18.0b,B 118.8 ± 27.9b,B 399.6 ± 57.2c,B 165.0 ± 52.6a,B 140.6 ± 15.7ab,B 98.2 ± 15.5b,B 391.6 ± 61.8c,B 190.2 ± 46.9a,B 66.6 ± 9.0b,C 66.3 ± 35.5b,B 314.4 ± 51.3c,C

596.0 ± 167.4a,A 445.7 ± 285.2a,A 339.2 ± 81.3a,A 416.3 ± 117.3a,A 165.5 ± 57.2a,B 234.1 ± 89.3a,AB 224.4 ± 50.7a,B 332.1 ± 27.4b,A 171.2 ± 9.6a,B 233.5 ± 39.3b,AB 224.9 ± 32.4b,B 356.5 ± 22.5c,A 120.0 ± 13.2a,B 133.5 ± 44.1a,B 100.8 ± 30.0a,C 336.5 ± 63.5b,A

80.6 ± 36.7a,A 130.9 ± 40.1b,A 101.8 ± 12.4ab,A 78.8 ± 24.2a,A 95.1 ± 16.2a,A 117.7 ± 29.1a,A 105.3 ± 16.7a,A 113.4 ± 22.7a,B 82.6 ± 24.9a,A 63.9 ± 30.7a,B 102.2 ± 31.8a,A 63.6 ± 20.5a,AC 78.2 ± 16.2a,A 62.9 ± 11.6ab,B 85.5 ± 27.2a,A 41.8 ± 4.9b,C

32.4 ± 2.1a,AB 30.9 ± 1.0a,A 41.5 ± 1.3b,A 38.3 ± 2.9c,A 28.6 ± 0.2a,A 49.4 ± 2.3b,B 56.0 ± 1.9c,B 45.5 ± 4.1b,B 37.4 ± 2.9a,C 34.9 ± 3.3a,A 44.5 ± 2.5b,A 43.6 ± 3.8b,B 34.1 ± 3.4a,BC 21.5 ± 7.7b,C 13.2 ± 2.7c,C 34.6 ± 2.0a,A

39.6 ± 3.0a,A 33.1 ± 2.1b,A 40.8 ± 1.7a,A 32.1 ± 2.2b,A 29.0 ± 0.5a,B 34.6 ± 3.5b,A 28.6 ± 0.7a,B 37.8 ± 4.0b,A 27.0 ± 1.3a,B 33.9 ± 4.0bc,A 32.0 ± 1.9ab,C 39.2 ± 5.3c,A 34.7 ± 1.5a,C 24.1 ± 3.8b,B 29.0 ± 1.3ab,B 32.4 ± 10.1ab,A

131.3 ± 3.4a,A 135.7 ± 3.8a,A 149.6 ± 6.0ab,A 177.1 ± 49.0b,A 104.4 ± 3.8a,B 127.3 ± 10.7b,A 124.0 ± 5.7b,A 177.3 ± 17.8c,A 105.5 ± 2.8a,B 113.2 ± 8.6a,B 138.7 ± 36.4ab,A 172.1 ± 48.9b,A 93.5 ± 2.7a,C 81.2 ± 6.2a,C 81.5 ± 7.2a,B 134.3 ± 18.2b,A

TF BCFR 0.49 0.49 0.75 0.54 1.12 0.53 0.53 1.20 0.96 0.60 0.44 1.10 1.59 0.50 0.66 0.93

0.44 0.40 0.31 0.43 0.43 0.54 0.52 0.75 0.70 0.95 0.94 1.35 0.78 0.82 0.67 2.68

Values followed by the same letter did not differ; small letters showed differences between amendment effects within the same site (P ≤ 0.05, LSD), capital letters demonstrated the difference between sites of plant grown in the same treatment (P ≤ 0.05, LSD). Data was represented as means ± standard deviation (n = 4).

When the Cd/Zn contaminated paddy soils were compared with the agricultural soil used in this study, it is apparent that they were relatively similar in many chemical properties such as pH (in the neutral range), organic matter (2–3%), and EC (0.11–0.13 ds m−1). However, the CEC in agricultural soil was much higher than in contaminated soils. In addition, the concentrations of some elements (P, Mg, K) were much lower in Cd/Zn contaminated soils. The present study also analyzed the chemical properties of soil additives used in the experiment including organic additives (cow manure and rice straw) and inorganic additives (dicalcium phosphate and zeolite). Both cow manure and rice straw had higher concentrations of organic matter, extractable Si (data no shown) and high EC values (3.6–4.3 ds m−1) than the inorganic additives. Effects of Amendments on Heavy Metal Availability The results of the pot study demonstrated the effectiveness of inorganic additives in reducing the bioavailability of Cd and Zn. The application of 5% dicalcium phosphate lowered the concentration of available Cd by 45% while 5% zeolite decreased the concentration of available Zn by 17%. Phosphorus (P) is commonly added to soils in order to reduce the availability of heavy metals including Cd (Huang et al. 2012). This is probably due to phosphate-induced Cd2+ adsorption and precipitation of Cd as Cd(OH)2 and Cd3 (PO4 )2 with the addition of KH2 PO4 (Bolan et al. 2003). Li et al. (2008) indicated that the properties of inorganic amendments as well as the undergoing chemical reaction were related to the increase in the available heavy metals in the soil. While zeolite had no effect on Cd availability, it could decrease Zn availability. Zeolite had a much higher CEC (137.4 cmol kg−1) compared to

dicalcium phosphate (8 cmol kg−1) and cow manure (42 cmol kg−1), indicating its high ability to exchange cations such as Zn. Hence, it is more effective than cow manure and dicalcium phosphate in Zn immobilization. Effects of Amendments on the Growth of Maize All amendments in this study other than zeolite showed some influence on the growth of maize both in the pot and field trial experiments. In the pot study, the biomass production of different amendments (two rates of treatment) followed the order: 12.5% rice straw >20% cow manure >5% dicalcium phosphate ∼10% cow manure ∼6.25% rice straw. In the field study, 20% cow manure was most effective in increasing growth at the control site while both rice straw (12.5%) and cow manure additions resulted in the highest biomass production in maize grown at the site with low Cd. There was no significant change in growth (compared to plants grown on non-amended soil) of plants grown at sites with high and moderate Cd concentrations. Organic additives such as cow manure, pig manure, and manure compost have been shown to promote plant biomass production especially in crop plants such as mustard, rye, oat, radish, corn, basil (Clemente et al. 2003, 2005; Putwattana et al. 2010; Chaiyarat et al. 2011). The present study also demonstrated higher biomass production in maize treated with cow manure and rice straw. Both soil additives had high organic matter (OM) contents and high silicate concentration. OM acts as a nutrient pool, increases CEC and buffer capacity and improves soil physical properties by reducing compaction of soil (Stewart et al. 2000). In addition, OM reduced the bioavailability of Cd, alleviating its toxicity resulting in enhanced biomass production (Li et al. 2008).

172 There is increasing evidence that silicon (Si) is beneficial for the healthy growth of plants such as rice (Liang et al. 1994; Ma and Yamaji 2006). The addition of Si reversed the growth inhibition induced by Cd toxicity (Shi et al. 2005), i.e., alleviating the toxic effect of Cd (Liang et al. 2005). The alleviation effect was attributed not only to the immobilization of Cd, but also to a Si-mediated mechanism in plant such as stimulation of antioxidant systems, alleviation of photosynthesis inhibition, and detoxification of metals within plants (Neumann and zur Nieden 2001; Shi et al. 2005; Liang et al. 2007). However, the mechanism of detoxification is not fully clear (Gu et al. 2011). Effects of Amendments on the Uptake of Cd and Zn The addition of cow manure, dicalcium phosphate, and rice straw resulted in the decreased concentrations of Cd and Zn in maize especially in the aboveground parts, both in the pot and field trial experiments. The decrease of Cd and Zn mobility in the soil influenced the transfer of these metals into generative organs of the plants (grain) to a slight extent. Several studies have demonstrated that organic amendments (cow manure and rice straw) which contain high organic matter, could decrease Cd accumulation in crop plants, including wheat (Narwal and Singh 1998), African basil (Chaiyarat et al. 2011), rice (Li et al. 2008), sorghum, and sunflowers (Luca et al. 2007). Similarly, lowered Zn concentrations in plants (e.g., alfalfa and Indian mustard) were observed when some compost and manure were added (Miller et al. 1995). The addition of manure, however apparently caused an increase in Cd and Zn accumulation in wheat, ryegrass, and basil (Alm˚as and Singh 2001; Narwal and Singh 1998; Putwattana et al. 2010). The efficiency of heavy metal immobilization in soil on reducing their plant uptake probably depends on both the species of crop and the soil characteristics. Distribution of heavy metals in soils is influenced by total metal concentration, pH, organic matter content (OM) and oxidation-reduction potential (Meers et al. 2005; Rodr´ıguezMoroto et al. 2003). Organic matter and pH are two most important soil factors that control Cd availability (Amini et al. 2005). As pH decreases, the amount of Cd in the plants increases (Sappin-Didier et al. 2005). OM can decrease bioavailability of heavy metals in soil due to the ability of OM to redistribute heavy metals from soluble and exchangeable forms to unavailable forms such as fractions associated with organic materials, carbonates, or metal oxides (Walker et al. 2004). In this study, it was observed that pH and total metal contents were not consistent predictors of metal bioavailability since they were not significantly changed after 3 months. Thus, the quality of soil OM may play an important role for ability to bind and accumulate Cd. There have been very few studies on the effects of rice straw on heavy metal accumulation in plants. The application of rice straw to Cd/Zn soils in our study significantly reduced Cd and Zn accumulation in the shoots of maize. The efficiency of both cow manure and rice straw in decreasing the Cd and Zn accumulation in maize shoot could be attributed to their high Si contents. Silicate fertilizer decreased Cd accumulation in shoots of rice (Liang et al. 2005; Gu et al. 2011), basil (Putwattana et al. 2010), maize (da Cunha and do Nascrimento 2009). Si can coprecipitate with heavy metals and block

N. Putwattana et al. metal transfer in plants (Gu et al. 2011). In addition, heavy metals deposited with Si in the root endodermis can physically block apoplast bypass flow across the roots (Shi et al. 2005). Cd and Zn uptake by maize in this study was also reduced by the addition of dicalcium phosphate. The application of P or phosphates in various forms such as NaH2 Po4 , NH4 H2 PO4 , rock phosphate, superphosphate KH2 PO4 , Ca(H2 PO4 )2 , have been shown to decrease Cd and Zn concentrations in shoots of cabbage (Wang et al. 2008), Spinacea oleracea (Dheri et al. 2007), and Sedum alfredii (Huang et al. 2012). Cd and Zn phytoavailability may be reduced through a combination of several mechanisms such as the formation of mixed–metal phosphates, enhancement of sorption mechanisms such as surface complexation, and ion exchange (Cao et al. 2004; Wang et al. 2008). Cd and Zn Accumulation in Maize Grain In Mae Sod district, maize grains are used for animal feed and cobs are sold to the dyeing industry to be used as fuel. Although the soils in Padae village were seriously contaminated by Cd and Zn, the concentrations of Cd in maize grain (0.1 mg kg−1) did not exceed 0.5 mg kg−1, a tolerable level recommended for livestock consumption (Chaney 1989), or 0.2 mg kg−1, the EU threshold value for feed materials. However, there was no standard limit for Zn in animal feed. But based on the tolerance limit for Zn in foods in China of 50 mg kg−1(Guo et al. 2011), the Zn concentrations in maize grain (27–39.6 mg kg−1) were still within accepted limits. The application of various soil additives (cow manure, dicalcium phosphate, rice straw), yielded Cd and Zn concentrations in grain below the tolerance limits in food or animal feed. All amendments decreased Cd and Zn concentrations in shoots of maize grown in paddy soil with the highest concentrations of Cd (36.5 mg kg−1) and Zn (1520.8 mg kg−1). Lime, fly ash, and steel slag have all been shown to be effective in reducing Cd concentrations in rice or maize grains grown in Cd or Zn contaminated soils (Li et al. 2008; Gu et al. 2011; Guo et al. 2011). The cost of some pure amendments (e.g., zeolite, dicalcium phosphate, hydroxyapatite) is relatively high. Since cow manure and rice straw are organic by-products of low cost and easy to obtain, they may prove to be lower cost substitutes. The results of this research demonstrated that under realistic field conditions soils of neutral pH (6.9–7.4), with Cd and Zn concentrations as high as 37 mg kg−1 and 1521 mg kg−1, respectively, do not cause excessive uptake of Cd and Zn by maize and, accordingly, are not hazardous to the food chain. However, under field conditions, one also has to consider the influence of decomposing humic material, which may interact with clay particles and alter the CEC and pH of a soil (Evangelou et al. 2004). In addition, periodic drying of the soil can influence the precipitation of metal oxides (Adam and Anderson 1983).

Acknowledgments The authors are grateful to Assist. Prof. Philip D. Round for editing the manuscript.

Maize Growth and Uptake of Cd and Zn from Paddy Soils Funding This research was supported by the grants from the Center of Excellence for Environmental Health, Toxicology and Management of Chemicals Under Science & Technology Postgraduate Education and Research Development Office (PERDO); and the National Research Council of Thailand.

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