Fly ash application in nutrient poor agriculture soils: Impact on methanotrophs population dynamics and paddy yields

Ecotoxicology and Environmental Safety 89 (2013) 43–51

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Fly ash application in nutrient poor agriculture soils: Impact on methanotrophs population dynamics and paddy yields Jay Shankar Singh a,n, Vimal Chandra Pandey b a b

Department of Environmental Microbiology, Babasaheb Bhimrao Ambedkar (Central) University, Raibarelly Road, Lucknow-226025, Uttar Pradesh, India Department of Environmental Science, Babasaheb Bhimrao Ambedkar (Central) University, Raibarelly Road, Lucknow-226025, Uttar Pradesh, India

a r t i c l e i n f o

abstract

Article history: Received 8 September 2012 Received in revised form 7 November 2012 Accepted 9 November 2012 Available online 20 December 2012

There are reports that the application of fly ash, compost and press mud or a combination thereof, improves plant growth, soil microbial communities etc. Also, fly ash in combination with farmyard manure or other organic amendments improves soil physico-chemical characteristics, rice yield and microbial processes in paddy fields. However, the knowledge about the impact of fly ash inputs alone or in combination with other organic amendments on soil methanotrophs number in paddy soils is almost lacking. We hypothesized that fly ash application at lower doses in paddy agriculture soil could be a potential amendment to elevate the paddy yields and methanotrophs number. Here we demonstrate the impact of fly ash and press mud inputs on number of methanotrophs, antioxidants, antioxidative enzymatic activities and paddy yields at agriculture farm. The impact of amendments was significant for methanotrophs number, heavy metal concentration, antioxidant contents, antioxidant enzymatic activities and paddy yields. A negative correlation was existed between higher doses of fly ashtreatments and methanotrophs number (R2 ¼ 0.833). The content of antioxidants and enzymatic activities in leaves of higher doses fly ash-treated rice plants increased in response to stresses due to heavy metal toxicity, which was negatively correlated with rice grain yield (R2 ¼ 0.944) and paddy straw yield (R2 ¼ 0.934). A positive correlation was noted between heavy metals concentrations and different antioxidant and enzymatic activities across different fly ash treated plots.The data of this study indicate that heavy metal toxicity of fly ash may cause oxidative stress in the paddy crop and the antioxidants and related enzymes could play a defensive role against phytotoxic damages. We concluded that fly ash at lower doses with press mud seems to offer the potential amendments to improving soil methanotrophs population and paddy crop yields for the nutrient poor agriculture soils. & 2012 Elsevier Inc. All rights reserved.

Keywords: Antioxidants Fly ash Heavy metals Methanotrophic bacteria Paddy crop

1. Introduction The rise in demand for power in domestic, agricultural and industrial sectors has increased the pressure on coal combustion and thus aggravated problem of fly ash (FA) generation/disposal. According to current estimates, the FA production may increase to 170 million tons yr  1 by 2012, and 225 million tons by 2017 (Singh, 2012). Therefore, FA management remains the great

Abbreviations: ANOVA, Analysis of variance; APX, Ascorbate peroxidase; CAT, Catalase; DAS, Day after sowing; EDTA, Ethylene di-amine tetra acetic acid; EC, Electrical conductivity; FA, Fly ash; FYM, Farm yard manure; GSSG, Glutathione disulfide; NADPH, Nicotinamide adenine dinucleotide phosphate; MB, Methanotrophic bacteria; MMO, Methane mono-oxygenase; PM, Press mud; ROS, Reactive oxygen species; SPSS, Statistical package for the social sciences; SM, Soil moisture; NTPP, National thermal power plant; MPN, Most probable number; PVP, Poly vinyl pyrrolidone; GR, Glutathione reductase n Corresponding author. E-mail addresses: [email protected] (J.S. Singh), [email protected] (V.C. Pandey). 0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.11.011

concern of the current century. FA contains traces of toxic elements and heavy metals (Pandey and Singh, 2010), but also has some macro- and micro-nutrients and thus can be used as soil amendments/conditioner to boost the soil health and crop productivity (Singh et al., 2011). Toxic effect of FA is insignificant, and concentration of toxic elements within permissible limits at its low doses in some plantation work (Pandey et al., 2009). Hence, major initiatives have been taken in India and elsewhere to use such a cost effective resource in large volumes in agriculture (Lee et al., 2007; Pandey and Singh, 2010; Singh et al., 2011). The Indian subcontinent has been affected worst by humans since long. This could be the reasons climate formations being altered and/or destroyed for paddy agriculture and even other similar purposes in the region. In the dry tropical region, low soil moisture status due to scanty rainfall and higher temperature affects soil functioning and paddy crop productivity (Singh et al., 2011). The soils of most of the dry-land rice agro-ecosystem are nutrient poor (Singh et al., 2010). It is here that micro-elements in the FA and press mud could be crucial to paddy crop productivity.

J.S. Singh, V.C. Pandey / Ecotoxicology and Environmental Safety 89 (2013) 43–51

experimental proofs for influence of FA and organic manure application on MB population in faddy fields are lacking. Further, it is still to be ascertained whether the FA application alters the population structure of the MB. Therefore, estimates of the methanotrophic population in paddy agriculture soil could be the effective means of assessing the impact of FA inputs on rice productivity and so also the abundance of MB following such treatments. The most important attribute, which makes FA and PM appropriate for farming, is its texture and the fact that it contains almost all the essential crop plant nutrients, the amendments improves the soil physico-chemical properties and microbial diversity (Kohli and Goyal, 2010; Singh et al., 2011). In view of this, it seemed important to assess the role of FA and PM amendments on soil physico-chemical properties, paddy crop yield and the methanotrophic population. Keeping in view the implication of the FA application on the improvement of various soil microbial properties and crop yield, an experiments was conducted on dry land paddy field, with the objectives: (a) to assess the effect of FA and PM inputs on MB population, heavy metal contents, antioxidants, antioxidative enzymatic activities, paddy yields and, (b) to examine the statistical correlation between treatments, MB population and paddy crop productivity.

2. Materials and methods 2.1. Experimental sites and climate This study was conducted at the agriculture farm (261520 2100 N; 801570 2000 E; 110 m msL) of the Department of Environmental Science, Babasaheb Bhimrao Ambedkar (Central) University, Lucknow, Uttar Pradesh, India. The soil was slightly alkaline, sandy loam, nutrient poor with moderate water holding capacity and grey in colour (Singh et al., 2010). Lucknow has a hot sub-tropical climate with warm summers and cool, dry winters. Summers (April to May) are quite hot with the temperature reaching 45 1C. Winters (December to February) are relatively cool with the maximum temperature 21 1C and minimum as low as 4 1C or even less. Fog formation is very often during the winter. The average annual rainfall ranged from 900 to 1100 mm during wet season of late June to October. However, during the last few years, it is extremely variable and random and at times, causing drought spells of varying degree and duration. The average monthly temperature and rainfall for the study area are presented in Fig. 1. 2.2. Set-up of experimental plots for paddy crop cultivation Field experiments were conducted in the rainy paddy crop season (July to November 2010) adopting a high-yielding rice (Oryza sativa) variety HUBR 2–1 (Malviya Basmati Dhan 1). The rice variety is semi-dwarf, with stiff stems, has fairly strong tillering ability, and tolerant to several paddy diseases such as blast, bacterial leaf blight, stem borer etc. The dry-land rice variety used presently was from the Department of Genetics and Plant Breeding, Institute of Agriculture Sciences, Banaras Hindu University, Varanasi. The methods for the preparation of experimental plots were according to our earlier investigations performed in the same area (Singh et al., 2010).

Rainfall

Max temp

Min temp

350

45

300

40 35

250

30

200

25

150

20 15

100

10 50

5

0

0 Jan Feb Mar Apr May Jun

Jul Aug Sep Oct Nov Dec

Fig. 1. Metrological data of study area during the year 2010.

Temperature ( C)

For achieving maximum crop productivity the importance has been attached to the application of FA, press mud (PM), farm yard manure (FYM) and other organic manures to conserve soil moisture and improve fertility of the nutrient poor agriculture soils (Odlare and Pell, 2009; Pandey and Singh, 2010). Paddy is an important crop of Indian agriculture. Owing to the shrinking cultivable land resources, and the demand to produce more and more food per unit area, has made agriculture heavily dependent on chemical fertilizers. The indiscriminate use of chemical fertilizers affects soil health and, leads to a negative impact on soil productivity by eliminating diverse types of beneficial microorganisms such as methanotrophic bacteria (MB) (Singh et al., 2010). Thus for sustainable paddy agriculture, all our efforts should be streamlined to protect and sustain soil health. In this context, that FA amendment is gaining importance in rice agriculture (Singh et al., 2011). FA contains heavy metals such as Cu, Ni, Cr, Cd, etc., that exhibit metal toxicity in plants (Lee et al., 2007; Pandey and Singh, 2010). These metals at supra-optimal condition become phytotoxic due generation of reactive oxygen species (ROS) and affect growth, development, and yield of the plants (Pandey et al., 2010). It is well known that heavy metals particularly redox metals may provoke oxidative stress with over production of ROS such as superoxide radicals (O2 ), hydroxyl radicals (OH  ), hydrogen peroxide (H2O2) etc., (Foyer et al., 1997). The ROS react very rapidly with DNA, lipids and proteins causes the plant cell damage (Navari-Lazzo and Quartacci, 2001). The tolerance capacity of plants to heavy metals depends on an interrelated network of physiological and molecular mechanisms (Bah et al., 2011). One of the mechanisms that make a plant species tolerant to heavy metal stress is the presence of strong antioxidant defence system (Pandey et al., 2010). In response to oxidative stress due to heavy metal toxicity the plants produces antioxidants to detoxify ROS that includes carotenoids, ascorbate, glutathione, tocopherols, anthocyanins and antioxidants enzymes such as superoxide dismutase, catalase, glutathione peroxidise, peroxidase, as well as enzymes involved in the different antioxidants enzymatic cycles (Bah et al., 2011; Pandey et al., 2010; Upadhyay et al., 2012). Although, a number of experiments have been conducted to demonstrate the application of FA as a soil amender to enhance the crop productivity (Pandey et al., 2009, 2010; Singh et al., 2011), but the information regarding the antioxidative defense response in the paddy crop amended with FA is still in incipient stage. Therefore, a precise knowledge would be useful about the change in Cu, Ni, Cr and Cd, induced oxidant stress and enzymatic antioxidant system in rice plants. FA is recognized as the useful resource and not just a waste, and could be the potential inorganic soil amendment to raise rice productivity and also to restore the soil nutrient balance in paddy soils (Lee et al., 2007). There are reports that the application of FA, compost and PM or a combination thereof, improves plant growth, soil microbial communities and their activities (Bougnom et al., 2010). Also, FA in combination with FYM or other organic amendments improves soil physico-chemical characteristics, rice yield and microbial processes in paddy fields (Pandey and Singh, 2010; Singh et al., 2011). However, the knowledge about the impact of FA inputs alone or in combination with other organic amendments on soil methanotrophic bacteria (MB) population in paddy soils is almost lacking. MB are the only known biological sink for the potent greenhouse gas methane (Singh et al., 2010). Therefore, population size of MB in the paddy agro-ecosystem soil is the important factor in influencing the regional/global methane oxidation. With the FA input in soils, the physical and chemical changes within the soil first may affect the microbial communities including MB as the latter are the first to be exposed to the soil changes. However, the

Rainfall (mm)

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J.S. Singh, V.C. Pandey / Ecotoxicology and Environmental Safety 89 (2013) 43–51 The experimental plots (each having 6  3 m size) were arranged in a completely randomized block design (RBD) with three replicates. To divide each experimental plot a strip of 30 cm was made to avoid the possibility of nutrients and microbial exchange in soils among the experimental plots. The various treatments applied were, control (no amendments), press mud (PM) (10 t ha  1) þ fly ash (FA) (10 t ha  1), PM (10 t ha  1) þ FA (50 t ha  1) and PM (10 t ha  1) þ FA (100 t ha  1). In each of the given FA and PM treatment combinations, the PM amendment was added at a fixed rate of 10 t ha  1. A blanket application of NPK fertilizer (160:80:40 kg ha  1) served as the basal dose. Half of the suggested dose of N was applied, at the time of seed sowing (July) and panicle initiation (September). The FA was obtained from National Thermal Power Plant (NTPP), Unchahar, Uttar Pradesh and PM (organic manure with mild acidic features) from the Department of Applied Plant Sciences, B.B. Ambedkar (Central) University, Lucknow. The FA and PM treatments were applied during mid July 2010 according to Schutter and Fuhrmann (2001). The status of heavy metals and macro- and micro-elemental composition of FA applied in this study are already described (Singh et al., 2011). The rice seed sowing, experimental site maintenance, crop harvesting (November) and estimation of selected parameters (rice gain and paddy straw yields) for paddy crop productivity were done according to Singh et al. (2010). 2.3. Soil sampling, soil analyses and enumeration of viable soil methanotrophs From each experimental plot, soil samples were collected every month during the crop cycle (July to November) 2010 from the 0 to 15 cm depth, 20 days after application of treatments. From each experimental plot, 10–15 soil cores (10–15, 2.5 cm diameter) were sampled randomly, and combined as one composite sample after thorough mixing by hand. The composite samples (field moist condition) were sieved (2 mm mesh), divided into two equal halves, and stored at 4 1C for further analysis. One part (in triplicate) analysed for physico-chemical parameters (only once at the beginning of the study period), and the second part (in triplicate) used for inorganic-N (NH4þ –NþNO3 –N). FA, PM, metals and micro-elements were analyzed according to using a Varian Atomic Absorption Spectrometer (AA240FS). Soil sub-samples (about 10 g) were dried at 105 1C for 24 h (to a constant weight) for the gravimetric soil moisture content (percent on dry soil basis). Soil pH was measured in de-ionized water by glass electrode, after shaking (1 h) on a gyratory shaker (1:2.5, soil: water). Soil inorganic –N (NH4þ –N þ NO3 –N) was determined according to the methods of Jackson (1958). Total –C was analyzed by dichromate oxidation and titration with ferrous ammonium sulphate (Walkley, 1947). Total –N was analysed by semi-micro-Kjeldahl digestion and total –P colorimetrically after HClO4 digestion (Jackson, 1958). The viable MB population was enumerated by the new most-probable-number (MPN) technique with slight modifications as described by Saitoh et al. (2002). In present study, culture tubes were used as the alternative to microtiter plates. This modified MPN technique offers not only the precise estimates of small population of methanotrophs in a paddy soil with no possibility of overestimation of methanotrophs population, but also requires lesser equipment, labour, and is superior to traditional MPN method (Saitoh et al., 2002). 2.4. Heavy metal analysis For heavy metal analysis, oven dried plant samples (root, shoot and seeds) as well as soil samples were homogenized by grinding in a stainless steel blinder and after sieving (2 mm mesh size screen) kept at room temperature for further analysis. Grinded samples (1 g) were digested with a mixture of nitric, sulphuric and perchloric acid (6:1:2 by volume) initially for 2 h at 100 1C and then the temperature was raised up to 120 1C. The digestion was carried out in 250 mL Pyrex digestion tubes until white residue was achieved, avoiding complete evaporation of acids. Digested plant material was diluted with 50 mL double de-ionized water and used for heavy metal analysis. The heavy metal analysis of the plant samples and soil samples were conducted using a Varian Atomic Absorption Spectrometer (AA240FS) according to Pandey et al. (2010). All the plant samples were analysed in triplicate. 2.5. Antioxidants and antioxidative enzymatic activity in rice plant At the time of the crop maturity, rice plant leaves were sampled in the first week of November-2010 for the purpose of analysis of different enzymatic

45

activities. The quantitative estimation of ascorbate and glutathione levels in rice plants was done in present study. Total carotenoids were determined by using acetone and petroleum ether as extracting solvents and measuring the absorbance at 450 nm. Fresh leaf tissue (0.5 g) were homogenized using the liquid N2 in QB buffer (100 mM potassium phosphate buffer, pH 7.8, 1 mM EDTA, 1 percent Triton X-100, 15 percent glycerol) (Ni et al., 1996) for the Ascorbate peroxidase (APX) and catalase (CAT) activities. The homogenate was supplemented with 50 mg of poly vinyl pyrrolidone (PVP) per gram of fresh tissue for the glutathione reductase (GR) assay. Crude homogenate was centrifuged at 15,000 rpm for 20 min at 4 1C, and the supernatant fractions were used for determination of CAT, APX and GR assays. CAT (EC 1.11.1.6) activity was assayed by monitoring the decomposition of H2O2 spectrophotometrically at 240 nm (Aebi, 1983). The CAT activity [U (mg protein)  1] was calculated using a molar absorption coefficient of 40 mM  1 cm  1 for H2O2. APX activity (EC 1.11.1.11) was determined by the method of Asada (1992) in fresh leaf samples of rice plant. The reaction was initiated by the addition of H2O2. The H2O2 dependent oxidation of ascorbate was followed by monitoring the decrease in absorbance at 290 nm. One unit of APX was the amount of enzyme that oxidized 1 m mol of ascorbate min  1 at room temperature. Peroxidase activity (U mg protein  1) was determined following the method described by Sinha et al. (2007). The reaction mixture contained 100 mM Trisbuffer (pH 7.0), 10 mM pyrogallol, and 5 mM H2O2. The reaction was started by adding 25 mL enzyme solution and stopped after 5 min incubated at 25.8 1C by adding 1.0 mL 2.5 N H2SO4. The amount of purpyrogallin formed was measured spectrophotometrically at 425 nm. For GR activity, the reaction was initiated by the addition of NADPH at 25 1C. The reaction mixture consisted 100 mM potassium phosphate buffer, pH 7.0, contained 1.0 mM EDTA, 150 mM NADPH, 500 mM GSSG, and enzyme extract. The GR activity [U (mg protein)  1] was determined by the oxidation of NADPH at 340 nm with a molar absorption coefficient for NADPH of 6.2 mM  1 cm  1 according to Nordhoff et al. (1993).

2.6. Statistical procedures One-way ANOVA at 95 percent probability level was applied to examine the impact of FA and PM amendments on selected physico-chemical soil properties, heavy metal concentration, antioxidants status and antioxidants enzymes activity, paddy crop yields and the viable population of soil MB. Regression analysis was performed between treatments applied and the MB population. Pearson’s correlation analysis was used to assess the significance of the interrelationships between the soil heavy metals and antioxidants levels and antioxidative enzymatic activities across different treatments. All statistical analyses were conducted using MS-Excel and SPSS (Version-14).

3. Results and discussion 3.1. Impact of FA and PM amendments on soil physico-chemical characteristics The rainfall and temperature of the study area for each sampling dates (July to October-2010) is given in Fig. 1. The temperature during the paddy crop cycle was maximum (1C) at the 20 DAS (Day After Sowing) and minimum (1C) for 110 DAS. The precipitation during different sampling dates varied from 22 to 110 mm (highest for 20 DAS), and averaged 48.3 mm from 20 to 110 DAS (July to October). The selected physico-chemical parameters of fly ash (FA) and press mud (PM) as amendments are presented in Table 1. As indicated, the FA applied had an alkaline pH (8.7), low total –C, total –N and P but high micro-elements (Fe, Mn, Ca, Mo and Ni) compared to PM. The FA used in this study, was alkaline with low total –C, N and P and high major micro-elemental composition

Table 1 Chemical composition of fly ash (FA) and press mud (PM) applied in present experiment. The given values are means of three replicates7 SE. Treatments

FA PM

Physico-chemical parameters

Micro-nutrients (%)

Total–C (%)

Total–N (%)

Total–P (%)

WHC (%)

pH

Bulk density (g cm  3)

Fe

Mn

Ca

Mo

1.37 0.57 1.97 0.65

0.87 0.08 1.27 0.13

0.09 70.01 0.14 70.06

45.3 71.07 48.1 71.09

8.7 7 0.56 4.7 7 0.27

1.8 7 0.18 1.4 7 0.16

0.39 7 0.09 0.28 7 0.02

0.09 70.06 0.01 70.05

0.95 70.03 0.49 70.06

0.38 70.07 0.16 70.02

46

J.S. Singh, V.C. Pandey / Ecotoxicology and Environmental Safety 89 (2013) 43–51

Table 2 Variation in soil physico-chemical characteristics due to different treatments and sampling dates. Given values are means of three replicates 7SE. However, the bold figures given in parenthesis for given parameters and treatments are the means 7SE across different sampling dates. Treatment combinations

Sampling dates

Control

20DAS 40DAS 60DAS 80DAS 110DAS

PM þ FA (10 t ha  1)

20DAS 40DAS 60DAS 80DAS 110DAS

PM þ FA (50 t ha  1)

20DAS 40DAS 60DAS 80DAS 110DAS

PM þ FA (100 t ha  1)

20DAS 40DAS 60DAS 80DAS 110DAS

ANOVAa

Parameters SM content (%)

EC (dS m  1)

pH

Inorganic-N (NH4þ –N þNO3 –N) (mg g  1 soil)

18.4 7 1.2 18.3 7 1.3 17.2 7 1.2 13.8 7 1.5 09.2 7 1.2 (15.38 71.7)nn 27.9 7 2.1 27.5 7 2.3 25.7 7 2.2 23.2 7 2.4 12.4 7 1.5 (23.34 72.8)nn 30.1 7 2.9 29.2 7 2.5 28.4 7 2.3 24.3 7 2.2 13.6 7 1.7 (25.12 73.0)nn 30.6 7 2.8 29.7 7 2.4 28.5 7 2.2 28.2 7 2.1 13.8 7 2.2(26.167 3.1)nn F ¼3.15; P¼ 0.000

2.27 0.8 2.47 0.8 2.47 0.7 2.47 0.6 2.57 0.9 (2.3 70.4)NS 3.57 0.2 3.57 0.3 3.57 0.2 3.67 0.4 3.67 0.5 (3.5 70.2)NS 6.27 0.6 6.37 0.7 6.37 0.6 6.57 0.9 6.67 0.7 (6.4 70.7)NS 6.87 0.5 6.97 0.6 6.97 0.8 7.17 0.6 7.27 0.8(6.9 7 0.7)NS F¼ 1419.4; P¼ 0.000

6.77 1.2 6.87 1.1 6.97 1.3 6.77 1.2 6.87 1.5 (6.7 70.3)NS 6.97 1.4 7.07 1.5 7.17 1.6 7.17 1.8 7.27 1.5 (7.0 70.5)NS 7.97 1.7 8.07 1.9 8.07 1.8 8.17 1.7 8.27 1.9 (8.0 70.5)NS 8.37 1.8 8.37 1.7 8.37 1.5 8.67 1.8 8.77 1.6(8.4 7 0.8)NS F¼ 516.5; P ¼0.000

33.4 71.4 32.1 71.2 32.07 1.2 31.2 71.4 26.4 71.2 (31.07 1.2)n 25.5 71,4 23.3 71.3 22.2 71.3 21.1 71.2 18.2 71.5 (22.07 1.2)nn 26.7 71.5 26.5 71.7 26.2 71.3 26.1 71.2 25.8 71.7 (26.37 0.9)n 26.9 71.2 26.8 71.4 26.6 71.3 26.4 71.1 26.1 71.2(26.57 1.2)n F¼ 18.0; P ¼0.003

NS¼ Not significant, PM, Press mud, FA, Fly ash, SM, Soil moisture, EC, Electrical conductivity. a

Total samples used for this analysis was N ¼20 (4 treatments  5 sampling days). P o0.05. nn Po 0.01. n

(Fe, Mn, Ca, Mo and Ni) relative to PM. The high levels of calcium oxide and hydroxide are found in FA may significantly affects the soil pH levels. In the present study the higher concentration of Ca in FA could one of the major reasons for higher pH due to high FA doses (Kumar et al., 2008; Singh et al., 2010). Compared to FA, PM had lower pH with significant amount of soil C, N and P to support paddy crop growth. The lower pH in PM counter balance the higher alkalinity of FA when applied in combination i.e., PMþFA (10 t ha  1) and consequently made it the most effective strategy. The soil physico-chemical characteristics of the experimental plots are presented in Table 2. The FA and PM amended plots exhibited higher average soil moisture (SM) content (23.34–26.16 percent), electrical conductivity (EC) (3.5–6.9 dS m  1), pH (7.0– 9.4) and inorganic –N (22.0–26.5 mg g  1 soil) than the control plots. ANOVA revealed significant differences (P ranged from 0.006 to 0.000) for soil physico-chemical properties following the amendments. However, only SM (Po0.01) and inorganic –N (Po0.01–0.05) differed significantly for the sampling days (Table 2). 3.2. Impact of FA and PM amendments on heavy metals, antioxidants and antioxidative enzymes Table 3 The results showed that the level of heavy metals (Ni, Cu, Cr and Cd) in the leaves and rice plant parts (root, shoot and seeds) in 100 t ha  1 FA treated plot was significantly high compared to control plot (Table 4 and Fig. 4). ANOVA revealed significant variations in Ni, Cu, Cr, and Cd concentrations in the plant parts (root, shoot and seeds) due to treatments (Table 5). The concentration of Ni, Cu, Cr, and Cd across different treatments ranged in root (6.4–15.2, 6.3–11.2, 3.1–11.2 and 1.8–8.0 mg g  1 dry weight, respectively), shoot (3.1–8.7, 2.5–6.5, 1.8–7.0 and 0.8–3.0 mg g  1 dry weight, respectively) and seeds (0.4–5.2, 0.3– 2.7, 0.5–2.0 and 0.07–1.2 mg g  1 dry weight, respectively) of rice

plants (Fig. 4). Variations in Ni, Cu, Cr, and Cd concentrations in root, shoot and seeds of rice plants due to different treatments were significant (Table 5). At lower treatments (10 t ha  1 FA), the level of metals in paddy seeds was found not significant to their respective control. ANOVA revealed that in all the plant parts (roots, leaves, and seeds) concentrations of all metals were significantly (Po0.001) higher in 50 t ha  1 FA and 100 t ha  1 FA than the control. The metal availability to plants is depended on its solubility and is generally govern by the soil pH levels. In this study it might be possible that at the pH in higher FA doses (i.e., 50 and 100 t ha  1) the large portion of heavy metals could be immobilized in plant parts (root shoot and seeds). In present study among different examined rice plant parts the level of all heavy metals was higher in roots than the shoots followed by seeds (Fig. 4). The data of present experiment showed that concentrations of metals in the soil and plant parts of paddy increase with the increase in FA doses. Pandey et al. (2010) also reported that the quantity of different heavy metals in chickpea plant parts increased with increase in soil FA percentage during a pot experiment. The accessibility, and accumulation of heavy metals by plants may depends on different edaphic factors such as pH, salinity, soil mineralogy, texture, metal speciation, humic substance status, organic chelators and presence of other metal contents (Pandey et al., 2010). In present study the differences noted in the metal concentration in the various parts of the rice plant could be due to different cellular mechanisms of bioaccumulation of metals that may control their translocation and partitioning in the plant systems as suggested by Sinha et al. (2007). The data of present study indicate that metal concentration was more in roots as compared to shoots, as roots act as a barrier against heavy metal translocation and this might be a potential heavy metal tolerance strategy in the roots (Ernst et al., 1992). Reduced accumulation of metals in the shoots could be due to sequestration of most of the metals in the vacuoles of the root

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Table 3 Impact of fly ash (FA) and press mud (PM) inputs on the population of methanotrophic bacteria and paddy productivity. Values are means of three replicates7 1SE. Figures given in parenthesis indicate percent increase in methanotrophic population and paddy crop yields over the control value. Study parameters

Methanotrophs number (  104 g  1 soil)a Paddy crop productivityb

Grain yield (kg ha

1 c

)

Paddy straw (kg ha  1)c

a b c

ANOVAa

Treatment combinations Control

PM þFA (10 t ha  1)

PMþ FA (50 t ha  1)

PM þFA (100 t ha  1)

23.4 7 6.1 – 1546 7 86.5 – 2642 7 28.8 –

53.0 7 11.5 (126) 3686 7 89.2 (138) 4728 7 100.8 (78.9)

29.4 76.1 (25.6) 1678 788.3 (8.5) 28047 22.5 (6.1)

25.2 7 6.1 (7.6) 1658 7 87.3 (7.2) 2687 7 30.7 (1.7)

F¼ 3.06; P¼ 0.058 F¼ 146.45; P ¼ 0.000 F¼ 335.27; P ¼ 0.000

Total samples used for this analysis was N ¼20 (5 sampling dates  4 treatments). Rice plants were sampled after crop maturity to measure the paddy yields and total samples used in this analysis was N ¼ 12(3 replicates  4 treatments). Five rice hills (in triplicates) were considered to find out the mean values.

Table 4 Effect of FA treatments on total heavy metal concentrations, antioxidants and antioxidative enzymes levels in rice plant leaves. Parameters

Control Heavy metals (mg g  1 dry soil)b Ni Cu Cr Cd

ANOVAa

Treatment combinations PM þFA (50 t ha  1)

PM þFA (100 t ha  1)

7.20 70.33 5.23 70.33 4.37 70.23 2.43 70.19

9.067 0.35 6.237 0.48 5.647 0.48 3.607 0.63

12.21 7 0.42 7.89 7 0.23 7.607 0.63 4.12 7 0.45

F¼ 9340.40; F¼ 2185.03; F¼ 4967.08; F¼ 1527.86;

Antioxidants (mg g  1 fresh weight)b Ascorbate 3.13 7 0.14 Glutathione 21.86 7 0.61 Carotenoids 0.32 7 0.008

4.20 70.17 31.56 70.27 0.46 70.008

5.437 0.08 38.007 0.26 0.847 0.01

8.26 7 0.08 56.43 7 0.13 0.937 0.01

F¼ 29.32; Po 0.001 F¼ 1563.65; Po 0.001 F¼ 572.04; P o 0.001

Antioxidant enzymes (U min  1 mg  1 fresh weight)b Catalase 2.28 7 0.003 Ascorbate peroxidase 7.34 7 0.04 Peroxidase 134.527 0.35 Glutathione reductase 6.73 7 0.01

5.30 70.03 11.4 70.07 248.3 70.58 5.45 70.11

6.447 0.006 19.61 7 0.17 378.117 0.87 3.507 0.02

a b

5.52 7 0.46 4.34 7 0.58 3.68 7 0.33 1.80 7 0.06

PMþ FA (10 t ha  1)

9.55 7 0.06 46.22 7 0.40 412.667 0.27 2.37 7 0.08

P o 0.001 Po 0.001 Po 0.001 Po 0.001

F¼ 5498.99; Po 0.001 F¼ 6035.97; Po 0.001 F¼ 49448.48; P o0.001 F¼ 753.24; Po 0.001

Total sample analysed was N ¼ 12 (3 replicates  4 treatments). At the time of the crop maturity, plant leaves were sampled for the purpose of analysis of heavy metal contents and enzymatic activities.

Table 5 The results of ANOVA exhibiting impact of treatments on heavy metal status on root, shoot and seeds of paddy crop. Total sample analysed was N ¼ 12 (3 replicates  4 treatments). This analysis was performed on the data presented in Fig. 4. Study parameters

Root Shoot Seeds

Heavy metals Ni

Cu

Cr

Cd

F ¼1521.16; P o 0.01 F ¼267.73; P o0.001 F ¼1100.13; Po 0.01

F ¼424.58; P o0.001 F ¼781.56; P o0.01 F ¼269.78; P o0.001

F ¼1573.42; P o0.002 F ¼2380.40; Po 0.01 F ¼13716; P o0.001

F ¼3937.70; P o0.01 F ¼543.30; Po 0.001 F ¼271.60; Po 0.01

cells to render it non-toxic, which may be a natural toxicity defensive mechanism of the plant systems (Shanker et al., 2005). In general, that result of present investigation demonstrate that the increasing FA doses may cause a progressive augment in the levels of different heavy metals in the roots, shoots and seeds of rice plants (Fig. 4A–D). Based on comparative studies of metal content in plant parts it has been suggested that the uptake, translocation and accumulation mechanisms of the plants differed for different heavy metals and for the plant species (Baker and Walker, 1990). Liu et al., (2004) reported that the metal tolerant plants accumulate the smallest proportion of the total metals form the soil and consequently the lowest metal levels was noted in the shoot. Heavy metal accumulation capability of the plants from the soil can be estimated by using bioconcentration factor (BCF), which is based on the ratio of metal concentration in the roots to that in soil (Pandey et al., 2010).

In the present study, the calculated BCF (data not shown) values of all the metals in plant parts are less than one (critical value) indicating the low level accumulation of metals from treatments to rice plants parts. The mean data of heavy metals in different plant parts were found under critical value, and is accordance with results of Pandey et al. (2010). As the data indicated that the antioxidants levels (ascorbate, glutathione and carotenoids) and antioxidative enzymatic activities (catalase, ascorbate peroxidise, peroxidise and glutathione reductase) was increased in leaves of rice plants with increase in FA concentrations (Table 4). Across different treatments in rice leaves the catalase, ascorbate peroxidise, peroxidise and glutathione reductase ranged, respectively, from 2.28 to 9.55, 7.34 to 46.22, 134.52 to 412.66 and 6.73 to 2.37 U min  1 mg  1 fresh weight. A significant difference in the levels of antioxidants and antioxidative enzymatic activities in leaves was noted due to

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J.S. Singh, V.C. Pandey / Ecotoxicology and Environmental Safety 89 (2013) 43–51

Table 6 Correlation between heavy metals and different antioxidant and enzymatic activities in paddy leaves across treated with fly ash. Total samples analysed was N¼ 12 (3 replicates  4 treatments). This correlation analysis is based on the data presented in Table 4. Study parameters

Ni

Cu

Cr

Cd

Ascorbate

Glutathione

Carotenoids

Catalase

Ascorbate peroxidase

Peroxidase

Cu Cr Cd Ascorbate Glutathione Carotenoids Catalase Ascorbate peroxidase Peroxidase Glutathione reductase

0.987nn 0.899nn 0.953nn 0.981nn 0.995nn 0.885nn 0.992nn 0.961nn 0.903nn 0.946nn

0.935nn 0.983nn 0.959nn 0.976nn 0.913nn 0.998nn 0.913nn 0.947nn 0.957nn

0.982nn 0.901nn 0.909nn 0.992nn 0.935nn 0.837nn 0.997nn 0.985nn

0.937nn 0.951nn 0.965nn 0.981nn 0.877nn 0.989nn 0.981nn

0.990nn 0.905nn 0.969nn 0.984nn 0.893nn 0.953nn

0.905nn 0.985nn 0.977nn 0.906nn 0.959nn

0.917nn 0.850nn 0.982nn 0.983nn

0.930nn 0.943nn 0.963nn

0.822nn 0.913nn

0.977nn

nn

Correlation is significant at the 0.01 level (2-tailed).

different FA amendments (Table 4). Pearson’s correlation analysis indicated that soil heavy metals were significantly correlated with antioxidants and antioxidative enzymatic activities of rice plant leaves treated with different doses of FA (Table 6). The level of both the antioxidant levels and related enzyme activities in the rice plant leaves elevated two and three times over controls at 50 t ha  1 and 100 t ha  1 FA treatments, respectively. Pandey et al. (2010) also suggested that antioxidants in plant samples of chickpea increase with increasing FA doses to combat stresses due FA heavy metals. Presence of elevated levels of heavy metals in higher FA doses may favours the generation of several oxidative radicals that may cause oxidative damages to plant cells (Choudhary et al., 2007). Plants possess defense systems to alleviate and renovate the damages caused by oxidative radicals (Pandey et al., 2010; Upadhyay et al., 2012). Increase in the activity of various antioxidative enzymes in plants may result in the antioxidants synthesis and ultimately confers the tolerance of plants against heavy metal toxicity. In general, the increase in the antioxidants levels and antioxidative enzyme activities in FA treated paddy crop was found to be directly proportional to the levels of heavy metals accumulation in the leaves of rice plants. The higher antioxidative oxidative enzymes activity under FA heavy metal stress is possibly a result of generation of oxidative radicals. These results of present study indicate that heavy metal of FA induces oxidative stress in rice plant and that enhanced activity of antioxidative enzymes could play defense apparatus against oxidative damages. 3.3. Impact of FA and PM amendments on paddy yields The data on paddy crop yield such as rice grain and paddy straw as affected by FA and PM treatments are shown in Table 3. The rice grain (1658–3686 kg ha  1) and paddy straw yield (2687–4728 kg ha  1) were high for all FAþPM amended plots compared to controls (Table 3). It is evident that for selected paddy yield parameters, the most effective treatment was the combination PMþFA (10 t ha  1) (138 percent enhancement in rice grain and 78.9 percent in paddy straw yield relative to controls) followed by PMþFA (50 t ha  1) and PMþFA (100 t ha  1). ANOVA indicated significant differences in rice grain (F¼146.45; N¼ 12; P¼0.000) and paddy straw yield (F¼335.27; N¼12; P¼0.000) due to various FA and PM amendments (Table 3). The regression analysis showed a logarithmic negative relationship for FA amendments with rice grain (R2 ¼0.9442) (Fig. 3B) and paddy straw yield (R2 ¼0.9345) (Fig. 3C). With this analysis an attempt was made to examine the influence of different FA doses on rice grain and paddy straw yield, and due to this the data of control treatment was not considered. The data show that the FA and PM amendment combinations resulted in high paddy grain and straw yield, and the variations in such parameters due to amendments, were significant (Table 3).

The most effective treatment noted for the paddy grain and straw yield as well was the combination of PMþFA (10 t ha  1) followed by PMþFA (50 t ha  1) and PMþ FA (100 t ha  1). The ample availability of soil inorganic –N nutrients, optimum pH and SM condition due to PMþFA (10 t ha  1) treatments, is suggested to be more efficient for the high paddy yields. Further, in such plots, the higher paddy yields could also be correlated with the enhanced availability and uptake of inorganic –N particularly the NH4þ –N by the paddy crop due to improved soil conditions (Singh et al., 2011). On the contrary, in PMþFA (50 t ha  1) and PMþFA (100 t ha  1) amended plots the efficiency of paddy crop productivity could have been suppressed because of the stressful soil conditions (high EC and pH ) at high FA doses. In the present study, a negative relationship between FA and PM treatments with rice grain yield (R2 ¼0.9442; Fig. 3B) and paddy straw yield (R2 ¼0.9345; Fig. 3C) probably explains the suppressive effect of higher doses of FA due to heavy metal toxicity. In general, the significant increase in the paddy crop productivity due to FA incorporation at lower dose (10 t ha  1) might be due to the cumulative effect of inorganic –N, soil moisture status and relevant soil microbial phenomena in the improved soil environment. In the present study, minimum paddy grain (7.2 percent) and straw yield (1.7 percent) occurred for the PMþFA combination (100 t ha  1). The inorganic –N concentrations (NH4þ – NþNO3 –N) in PMþFA (100 t ha  1) amended plot, indicates its higher concentration (Table 2), and this situation could have arisen because of the reduced uptake of nutrients available by paddy the crop following adverse effects of high FA doses, and consequently the reduced paddy yield might be expected for plots treated with PMþ FA combination (100 t ha  1). 3.4. Impact of FA and PM amendments on soil methanotrophic number Across all treatments and sampling dates during paddy crop cycle, the average viable soil MB population for treatments ranged from 23.4 to 53.0  104 g  1 dry soil (Table 3), and for sampling days (DAS), the values varied between 9.0 and 78.0  104 g  1 dry soil (Fig. 2). Among various amendments, the PMþFA (10 t ha  1) showed highest MB number (53.0  104 g  1 soil) compared to other treatments. The order of MB number and paddy yields along the experimental plots followed the sequence: PMþFA (10 t ha  1)-PMþFA (50 t ha  1)-PMþ FA (100 t ha  1)-control. The increase in number of methanotroph due to various FA and PM combinations over control plots, were 126, 25.6 and 7.6 percent in PM þFA (10 t ha  1), PMþ FA (50 t ha  1) and PM þFA (100 t ha  1), respectively. A significant difference in MB population was noted due to treatments (F¼3.06; N ¼20; P¼0.058). The number of MB showed a logarithmic negative relationship (R2 ¼0.8333) across the various FA doses (Fig. 3A).

J.S. Singh, V.C. Pandey / Ecotoxicology and Environmental Safety 89 (2013) 43–51

Viable methanotrophs population ( 104 g-1 soil)

The data on viable methanotrophic bacteria (MB) among different treatments (Table 3) and sampling days (Fig. 2) recorded high average MB population in FA and PM treated plots 90 80 70

Control PM+FA (10 t /ha) PM+FA (50 t/ha)

60 50 40 30

PM+FA (100 t/ha)

20 10 0

Control PM+FA (10 t /ha) PM+FA (50 t/ha) PM+FA (100 t/ha)

20DAS 10 38 21 12

40DAS 30 57 33 31

60DAS 36 78 43 38

80DAS 34 75 40 36

110DAS 7 17 10 9

Viable methanotrophs population ( 104 g-1 soil)

Fig. 2. Variation in population size of viable methanotroph population for different sampling days and fly ash (FA) and press mud (PM) treatments. Given values are means of three replicates 71SE.

90 80 70 60 50 40

30 20 10 0 0

20

40

60

80

100

120

0

20

40

60

80

100

120

Rice grain yield (kg ha-1)

4500 4000 3500 3000 2500 2000 1500 1000 500 0

Paddy straw yield (kg ha-1)

6000

5000 4000

49

(25.2  104–53.0  104 g  1 soil) compared to control (23.4  104 g  1 soil). In the present investigation, soil methanotrophic population (104 cells) as determined by the new MPN technique, is lower than the number (107–108 cells) as reported by Vishwakarma et al. (2009) in paddy fields. It may be expected that the changes in paddy soil environment due to FA and PM amendments play the dominant role in determining growth and multiplication of soil methanotroph and consequently, a significant difference in the microbial population existed. The present study indicated the most effective treatment combination for MB population was PMþFA (10 t ha  1) followed by PMþFA (50 t ha  1) and PM þFA (100 t ha  1), respectively. The low concentration of soil inorganic –N (NH4þ –NþNO3 –N) status, optimum pH and SM condition in PMþFA (10 t ha  1) treatment to others could be the major reasons for the high methanotrophic population. The information regarding the impact of FA amendments on methanotrophic activity or number is almost lacking. However, some reports show that application of FA at the rate of 10 t ha  1 was optimum for bacterial population, soil dehydrogenase activity and microbial biomass (Kohli and Goyal, 2010). FA added at levels exceeding 10 percent, resulted in a decline microbial activity (Kirk et al., 2005). The least MB number in control plots (23.4  104 g  1 soil) in the present study might be due to the higher concentrations of soil inorganic –N (NH4þ – NþNO3 –N) (Singh et al., 2010, 2011). The exact mechanism of inhibition of methanotrophic activity due to NH4þ –N is still not clear. However, some report indicate that inhibition of methane consuming methane monooxygenase (MMO) enzyme by NH4þ –N (a competitive inhibitor of MMO) can significantly suppress the activity of methanotrophs (Schnell and King, 1994). Further, the low number of methanotrophs due to high soil NH4þ –N contents in present study can be explained in the light of the observations of Bender and Conrad (1994) on the suppressive effect of NH4þ –N on MB number in paddy fields. Fig. 4 Among sampling days, the greatest population of MB in different experimental plots was observed on 60 DAS and lowest on 20 DAS (saturated soil condition) and 110 DAS (extreme low SM condition) (Fig. 2 and Table 2). Wilshusena et al. (2004) confirmed that oxygen concentration present in soil water plays very crucial role in the growth and multiplication of MB. The data of present study showed that the population size of methanotrophs in all the treatments was greatly reduced on 20 DAS and 110 DAS (Fig. 2). The decrease in population size of MB on 20 DAS (saturated soil condition due to heavy rainfall) in soils may perhaps due to cause of anoxic situation be able to disturb the methanotrophic physiology and survival. In present study, the increase in MB number due to various FA and PM treatment combinations were 126, 25.6 and 7.6, respectively for PMþFA (10 t ha  1), PMþFA (50 t ha  1) and PMþ FA (100 t ha  1) treated plots (Table 3). The negative relationships between higher FA doses and MB population (R2 ¼0.8333; Fig. 3A), certainly explains the inhibitory impact of higher amounts of FA on the MB numbers due to heavy metal toxicity.

3000

4. Conclusions

2000 1000 0 0

20

40 60 80 100 FA and PM treatments (t ha-1)

120

Fig. 3. Regression analysis of fly ash (FA) and press mud (PM) treatments with (A) viable MB population size, (B) rice grain yield and (C) paddy straw yield. Total samples used in this regression analysis was N¼ 9 (3 treatments  3 replicates). n Po 0.01; nPo 0.001.

This study suggests that FA and PM inputs had significant impact the soil MB population, paddy yields and enrichment of nutrient poor dry-land paddy soils. The results indicate that heavy metal toxicity at higher doses of FA may cause oxidative stress in the paddy crop and the antioxidants and related enzymes could play a defensive role against heavy metal toxicity damages. The alterations in soil properties subsequent to FA and PM amendments caused the variations in soil physico-chemical properties which in turn affected the soil MB abundance, soil

50

J.S. Singh, V.C. Pandey / Ecotoxicology and Environmental Safety 89 (2013) 43–51

14 12

18

Root

Shoot

Seed

Root

16

Shoot

Seed

Ni (µg g-1 dw)

Cu (µg g-1 dw)

14 10 8 6 4

12 10 8 6 4

2

2

0

0

Control

PM+FA (10 t /ha) PM+FA (50 t/ha) PM+FA (100 t/ha)

Control

PM+FA (10 t /ha) PM+FA (50 t/ha) PM+FA (100 t/ha)

Control

PM+FA (10 t /ha) PM+FA (50 t/ha) PM+FA (100 t/ha)

10

14

9

12 10

Cd (µg g-1 dw)

Cr (µg g-1 dw)

8

8 6 4

7

6 5 4 3 2

2

1

0

0

Control

PM+FA (10 t /ha) PM+FA (50 t/ha) PM+FA (100 t/ha)

Treatments

Treatments

Fig. 4. Heavy metals concentrations of (A) Cu, (B) Cr, (C) Ni and (D) Cd mg  1 g  1dry weight in paddy plant parts (root, leaves and seeds) treated with different fly ash doses. Given values are means of three replicates 7 1SE.

nutrient status and paddy yield. Thus, the study based on short term experimentations indicates an ample scope for utilization of FA in combination with organic manures to improve soil fertility, increase the MB population and also to augment paddy productivity in dry-tropical regions. It is expected that molecular microbial community analyses or even better, the functional key methanotrophic populations involved in the methane consumption process (i.e., methane-oxidation: MMO gene encoding methane monooxygenase enzymes i.e., particulate MMO and soluble MMO) could offer a still better insight to future explorations. Although FA is an input material for agriculture applications, the unhealthy heavy metal concentrations and other toxic elements for human consumption in the plant associated with the FA amendments are to be addressed in greater details. Also, the practical value and profitable use of FA in paddy soil or even other agriculture practices can only be feasible through further investigations in field conditions.

Acknowledgments The author is extremely thankful to Professor S.P. Singh, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi for editing the manuscript. The author is thankful to the Head, Department of Environmental Science, Babasaheb Bhimrao Ambedkar (Central) University, Lucknow for availing the infrastructure and other facilities during the experiment. I also wish to thank the Head, Department of Applied Plant Sciences of this University for their valuable suggestions in designing, preparation of the experimental plots and application of treatments. Department of Genetics and Plant Breeding, Institute of Agriculture Sciences, Banaras Hindu University is also thankfully acknowledged for their valuable suggestions for paddy crop cultivation and providing the rice seeds. Financial assistance given to

Dr. Jay Shankar Singh as a Senior Research Associate (Scientist’s Pool Scheme; SRA No: 13(8243–A)/2008/Pool) by the Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi is also gratefully acknowledged.

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