Effect of fly ash application on soil microbial response and heavy metalaccumulation in soil and rice plant

A.K. Nayak a,n, R. Raja a, K.S. Rao a, A.K. Shukla a,b, Sangita Mohanty a, Mohammad Shahid a,R. Tripathi a, B.B. Panda a, P. Bhattacharyya a, Anjani Kumar a, B. Lal a, S.K. Sethi a,C. Puri a, D. Nayak a, C.K. Swain a

a Central Rice Research Institute, Cuttack 753006, Odisha, Indiab AICRP (Micronutrients), IISS, Bhopal, India

a r t i c l e i n f o

Article history:Received 11 February 2014Received in revised form26 March 2014Accepted 27 March 2014

Keywords:Fly ashHeavy metalsHeterotrophsNitrifiersOxidizersSoil enzymes

a b s t r a c t

Fly ash (FA), a byproduct of coal combustion in thermal power plants, has been considered as aproblematic solid waste and its safe disposal is a cause of concern. Several studies proposed that FA canbe used as a soil additive; however its effect on microbial response, soil enzymatic activities and heavymetal accumulation in soil and grain of rice (cv. Naveen) to fly ash (FA) application was studied in a potexperiment during dry season 2011 in an Inceptisol. Fly ash was applied at a rate of zero per cent (FS),five per cent (FA5), ten per cent (FA10), twenty per cent (FA20), 40 per cent (FA40) and 100 per cent(FA100) on soil volume basis with nitrogen (N), phosphorus (P) and potassium (K) (40:20:20 mg N:P:K kg�1 soil) with six replications. Heavy metals contents in soil and plant parts were analysed afterharvest of crop. On the other hand, microbial population and soil enzymatic activities were analysed atpanicle initiation stage (PI, 65 days after transplanting) of rice. There was no significant change in theconcentration of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), cadmium (Cd) and chromium (Cr)with application of fly ash up to FA10. However, at FA100 there was significant increase of all metalsconcentration in soil than other treatments. Microorganisms differed in their response to the rate of FAapplication. Population of both fungi and actinomycetes decreased with the application of fly ash, whileaerobic heterotrophic bacterial population did not change significantly up to FA40. On the other hand,total microbial activity measured in terms of Fluorescein diacetate (FDA) assay, and denitrifiers showedan increased trend up to FA40. However, activities of both alkaline and acid phosphatase were decreasedwith the application of FA. Application of FA at lower levels (ten to twenty per cent on soil volume basis)in soil enhanced micronutrients content, microbial activities and crop yield.

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1. Introduction

Fly ash (FA), a byproduct of coal combustion in thermal powerplants, has been considered as a problematic solid waste and itssafe disposal is a cause of concern. FA being a coal combustionresidue shows a wide variation in their physico-chemical andmineralogical properties depending on the nature of parent coal,conditions of combustion, type of emission control devices,storage and handling methods (Jala and Goyal, 2006). The majormatrix elements in FA are Si, Al, and Fe together with significantpercentages of Ca, K, Na and Ti. Ca was found to be the dominantcation in FA followed by Mg, Na and K (Matti et al., 1990). Al in FAis mostly bound in insoluble aluminosilicate structures, whichconsiderably limits its biological toxicity. Fly ash contains

substantial quantities of trace metals (Cu, Zn, Mn, and Mo) andtoxic elements such as vanadium (V), selenium (Se), arsenic (As),boron (B), aluminium (Al), Cd, lead (Pb), mercury (Hg) and Cr(Gupta et al., 2002), and exhibits metal toxicity in plants (Pandeyet al., 2010). The pH of FA can vary from 4.5 to 12.0 dependinglargely on the sulphur content of the parent coal and the type ofcoal used for combustion affects the sulphur content of FA. Theadverse environmental impact of dumping FA is contamination ofsoil and water with toxic and radioactive elements, land degrada-tion and air pollution (Mallik, 2011).

The coal based thermal power plants, that constitute about 60–70 per cent of total power generation capacity in India, use mostlybituminous coal with low calorific value and high ash content (35–45 per cent), which lead to production of large volume of fly ash(112 mt) per year (Alam and Akhtar, 2011). In the current scenarioof national dependence on coal for power generation, this figuremay increase further in the years to come. Considering the highdisposal cost (Rs. 50–100/mt) of FA and large area of land required

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n Corresponding author. Fax: þ91 671 2367663.E-mail address: aknayak20@yahoo.com (A.K. Nayak).

Please cite this article as: Nayak, A.K., et al., Effect of fly ash application on soil microbial response and heavy metal accumulation in soiland rice plant. Ecotoxicol. Environ. Saf. (2014), http://dx.doi.org/10.1016/j.ecoenv.2014.03.033i

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for its disposal, it is imperative that the generated FA be utilized tothe maximum extent (Dhadse et al., 2008). Considerable researchhas been focused on management and alternate use of fly ash inmanufacture of cement, bricks, land filling, fertilizer fill, etc. Rice isan important crop of Indian agriculture grown on a variety of soilin different ecosystems. Due to ever increasing demand for foodand shrinking of cultivable land resources, there is a need toproduce more and more food per unit area which has madeagriculture heavily dependent on chemical fertilizers. The indis-criminate use of chemical fertilizers affects soil health and, leads toa negative impact on soil productivity by eliminating diverse typesof beneficial micro-organisms (Singh et al., 2010). In recent years,for sustainable productivity and improving soil health FA amend-ment is gaining importance in rice agriculture (Singh et al., 2011).Though, utilization of FA in agriculture is limited because of its lowN and P contents, low soil microbial activity, high pH (Wong andWong, 1989); there are some reports which mention potential useof FA as a soil ameliorant for improving physical properties of soil(Shen et al., 2008), as a liming material (Kumar and Singh, 2003)and a source of available plant micro- and macro-nutrients(Rautaray et al., 2003). Besides this fly ash is also used for landfilling in the low lying fields which are subsequently used foragriculture and forestry purposes, application at such higher ratemodify the soil and fly ash ratio and may have consequences onenzymatic activity and nitrogen transformation (Pati and Sahu,2004). Due to the low bulk density and nutrient contents,generally the FA is applied in huge quantities to the agriculturalfields. It has been reported that the application of FA from 10 to200 t ha�1 did not show toxicity effects to the rice plants andincreased the yields of paddy (Bhaskarachary et al., 2012; Lee et al.,2006). Most of the studies on fly ash are mostly focused on itsimpact on plant growth and productivity, heavy metal accumula-tion in plant and management practices to minimize the adverseimpacts of FA. However, the impact of FA on soil fertility, soilmicrobial/biochemical activity and soil nitrogen cycling is verylimited (Pandey and Singh, 2010). Therefore, there is a need tothoroughly test the extent of heavy metal accumulation in soil andits overall impact on soil biological health, rice plant growth andheavy metal contents in order to establish an eco-friendly FA dosefor safe soil amendment. Hence, the objectives of the presentstudy are i) to investigate the impact of fly ash amendment onmicrobial responses in soil by measuring selected microbialpopulations, processes, and enzyme activities., and ii) to assessthe extent of heavy metal accumulation in soil and rice plant dueto application of FA.

2. Methodology

2.1. Experimental setup

A pot culture experiment was conducted in the dry season of 2011 with rice (cv.Naveen) in the net house of Central Rice Research Institute, Cuttack, India. The soilused in the experiment was an Aeric Endoaquept. The fly ash was collected fromthe FA-dykes of Aarti Steel Plant, Athagarh, Odisha, India and this fly ash wasstabilized at 52 1C for 24 h to kill off any pathogens followed by drying at roomtemperature for one week. After the stabilization period, the mixtures were groundto pass through a 4 mm sieve to obtain homogeneous samples before mixing withthe soil.

The fly ash and time-zero soil samples were analysed for pH (1:2), electricalconductivity (1:2), organic matter content, percentages of sand, silt, and clay, andextractable elements (Table S1, in the Supplementary materials section) usingmethods as described by Jackson (1973). The soil was thoroughly mixed, dried,ground and sieved through a 2.0 mm sieve and filled in earthen pots of 40 cmheight and 30 cm upper diameter lined with polythene sheet along with FA andmade up to 10 kg. The levels of FA were zero per cent, five per cent, ten per cent,twenty per cent, 40 per cent and 100 per cent on soil volume basis which wererepresented as treatments FS, FA5, FA10, FA20, FA40 and FA100, respectively. Eachtreatment was replicated six times of which three pots were used for microbial

analysis at panicle initiation stage (65 days after transplanting) and remainingthree were taken up to harvest of the rice crop. Each pot was planted with 25 daysold single rice seedlings and applied with recommended dose of NPK (40:20:20 mgN:P:K kg�1 soil) to all the treatments. N was applied in two equal splits, one atbasal and other at 60 days after transplanting. Destructive soil sampling from threerandomly selected replicated pots of each treatment was done at panicle initiationstage for analysis of microbial parameters.

The soils collected from each pot were hand mixed, part of the soil sample wasair dried and ground to pass through a 2 mm sieve and analysed for heavy metals.The remainder of the samples was placed in a plastic bag and stored in arefrigerator at 4 1C prior to microbial and biochemical analysis. All microbiologicaland biochemical analyses were performed within 5 days of sampling. Leaf areaindex was measured at flowering stage. Leaf area was recorded by putting eachfresh leaves of one hill flat in a digital Leaf Area Meter (LI-3100, LiCor Inc., Lincoln,Nebraska). The area thus obtained was divided by the area of ground to get leafarea index. Observations on grain yield and other growth parameters wererecorded at physiological maturity stage and plant samples were collected foranalysis of heavy metals contents. Moisture percentage of the grains was deter-mined using Infrared moisture analyser and the grain yield was recorded atfourteen per cent moisture content.

2.2. Estimation of microbial populations

Aerobic heterotrophic bacteria were enumerated by plating soil dilutions toagar media in petridishes. The agar medium used contained dilute (1:100 fullstrength) trypticase soy broth (Difco), ten per cent soil extract (prepared asdescribed by Zuberer (1994)), and 1.5 per cent agar (difco). Plates were incubatedat 2872 1C for 72 h prior to enumeration. Ammonium oxidizers and dentrifierswere enumerated using the MPN techniques of Schmidt and Belser (1994) andTiedje (1994), respectively.

2.3. Measurement of soil enzyme activities

Knowing the sources of specific soil enzyme activities would greatly enhanceour understanding of which group(s) of organisms are directly accessing a givennutrient resource particularly N and P, thus providing greater insight into thepathways by which energy and nutrients flow through the soil food web. Activitiesof alkaline and acid phosphatase were determined spectrophotometrically bymeasuring intensity of yellow colour due to formation of p-nitrophenol from p-nitrophenyl phosphate (Tabatabai, 1994); urease enzyme activity was measured byDouglas and Bremner (1971) method which involves spectrophotometric estima-tion of unhydrolyzed urea after an incubation period. Fluorescein diacetate (FDA)hydrolysis assay was conducted spectrophotometrically by measuring formation offluorescein (fluorescent yellow–green) to assess the overall enzyme activity of totalmicrobial population (Adam and Duncan, 2001).

2.4. Nitrogen mineralization potential, nitrification potential and denitrificationactivity in soil

Nitrogen mineralization potential was determined by estimating the anaerobicproduction of ammonium (Bundy and Meisinger, 1994). Nitrification potential wasdetermined using an aerobic incubation procedure (Schmidt and Belser 1982) inwhich a solution containing 50 mg L�1 NH4–N was added to 100 g freshly collectedair dried soil contained in a glass beaker of 500 mL capacity and was kept in theincubator for a period of three weeks at 25 1C, another 100 g soil (without NH4–Nsolution) was incubated separately as control. Moisture content of soil wasmaintained at field capacity (by periodic weighing and offsetting the loss ofmoisture) throughout the incubation period. The amount of NO3–N content of soilat the beginning and end of incubation period was determined by extracting soilwith 2 M KCl followed by a steam distillation method, and increase in NO3–Nconcentration during the incubation period was expressed as nitrification potentialof soil.

Denitrification activity was determined following the procedure described bySmith and Tiedje (1979); 20 g of freshly collected homogenized soil sample fromeach treatment was placed in the conical flasks of 250 mL capacity. 10 mL ofdeionized water and 100 mg of chloramphenicol was added to the soil and contentwas mixed to get slurry. The flasks were made air tight by sealing the mouth with arubber septa through which the needle of syringe could pass, the headspace of theflask was evacuated and filled with O2 free N2 gas for three times to make thesample anaerobic and approximately ten per cent of the headspace was replacedwith acetylene gas (C2H2). 10 mL of a solution containing of 56 mg KNO3–N L�1 wasadded and the flasks were shaken for 30 min to establish equilibrium betweendissolved and gaseous N2O. Gas sampling from the headspace was done periodi-cally at 15, 30, 45 and 60 min. Gas samples were analysed in a gas chromatograph(Thermo CERES 800 plus GC) for determination of nitrous oxide using a Porapak Qcolumn and an electron capture detector (ECD).

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Please cite this article as: Nayak, A.K., et al., Effect of fly ash application on soil microbial response and heavy metal accumulation in soiland rice plant. Ecotoxicol. Environ. Saf. (2014), http://dx.doi.org/10.1016/j.ecoenv.2014.03.033i

2.5. Heavy metal analysis

Soil and fly ash samples were air dried and homogenized by grinding in astainless steel grinder and then passed through a 2 mm sieve to analyse heavymetals. Phytoavailable heavy metals contents of fly ash and soil were determinedfollowing a DTPA extraction technique (Lindsay and Norvell, 1978). The grainsamples were oven dried at 70 1C and ground in a mortar and a pestle and weredigested in tri-acid mixture (10:1:4, HNO3:H2SO4:HClO4 acids) and the metals weredetermined using an atomic absorption spectrophotometer (Varian SpectrAA55B).Limits of detection (LOD) of various heavy metals (Fe, Zn, Mn, Cu, Pb, Cd and Cr)were in the range of 0.01 to 0.05 mg L�1.

All laboratory glassware for heavy metals analysis was pre-soaked in 50 mg L�1

detergent solution for no less than 8 h and washed by tap water, then soaked in150 mL L�1 HNO3 solution over night, and rinsed by de-ionized water. Theindividual standards of heavy metals (Fe, Zn, Cu, Mn, Pb, Cd and Cr) were obtainedfrom Merck (Germany) and were used for calibration and quality assurance. Inorder to check the precision of the instrument, blank samples (samples withknown heavy metals) were run after the analysis of every set of samples.

2.6. Statistical analysis

Experimental data were obtained in triplicate and were compared by one wayanalysis of variance (ANOVA) and comparison of significance difference of treat-ment means at po0.05 was done by Duncan's multiple range test.

3. Results and discussion

3.1. Initial characteristics of soil and fly ash

Physico-chemical properties of FA and the experimental soilused in this study are shown in Table S1 (in the Supplementarymaterials section). The FA used in our study was slightly alkalinewith pH 7.7 and the experimental soil was acidic with pH 5.8. Theelectrical conductivity of fly ash was almost same as that of soil(Table S1). It is earlier reported that most of the Indian fly ash isalkaline due to low sulphur content of coal and presence ofhydroxides and carbonates of calcium and magnesium (Maitiand Nandhini, 2006; Singh et al., 2011). In the FA, total heavymetal contents were much higher as compared to the experi-mental soil, whereas in case of macronutrients, P and S contentswere more in FA, while N and K contents were more in theexperimental soil. The heavy metals contents of FA followed theorder Fe4Mn4Zn4Cu4Pb4Cr4Cd whereas in soil, the orderwas Fe4Mn4Zn4Pb4Cu4Cr4Cd. In FA total N was in traces,total P 0.05 per cent, total K 0.2 per cent, total S 0.8 per cent andcarbon was not detectable whereas in soil respective contentswere 0.06 per cent, 0.03 per cent, 0.1 per cent, 0.06 per cent and0.56 per cent. Though fly ash contains some essential plantnutrients such as P, Mg, S, K and Ca it is devoid of N, which isessential for the plants (Singh et al., 2011). However, there aresome ill effects of using fly ash because of its pozzolanic property,the presence of heavy metal, the lack of an appropriate dose forsoil and crops and environment impact (Jala and Goyal, 2006).

3.2. Plant growth in fly ash amended soil

Under stress condition, the commencement of floweringdelays, thereby delaying the maturity period of the crops. In thisstudy, the days to 50 per cent flowering of rice plant lengthen bysix and ten days in FA40 and FA100, respectively over control;however FA5, FA10 and FA20 recorded no significant difference.Height of plant, tiller number, leaf area index, number of panicles,length of panicles, number of grains per panicle as well as thegrain yield were significantly increased in pots treated up totwenty per cent FA (FA20) over control (Table S2, in the Supple-mentary materials section) beyond which they started decliningand FA100 recorded lowest value. All the growth parameters andthe yield in FA10 and FA20 did not differ significantly. The FA40

and FA100 recorded significantly lower yield than all the othertreatments. Significantly higher grain as well as straw yield of ricewas recorded with application of fly ash up to twenty per cent,however, fly ash addition higher than twenty per cent decreasedthe yield. Similar observations were reported by Pandey et al.(2010) who observed the negative effect of increased doses of flyash in chickpea.. Fly ash contain traces of N and no organic carbon(Singh et al., 2011), hence at higher dose (volume basis) whenadded to soil, causes dilution, additionally increases the heavymetal contents thereby decreases the crop yield. The soil whichinherently possesses plant nutrients, when replaced by fly ash in afixed volume the nutrient contents get reduced in the resultantsoil and fly ash mixture. Thus, fly ash can at best be used as asupplement to chemical fertilizer (Kumar et al., 1998). It is alsoreported that higher doses of ash (more than 50 per cent) reducesthe chlorophyll content and hence the growth due to the higherconcentration of heavy metals and metalloids causing nutritionalimbalance (Gupta et al., 2002). For low doses of ash, there is anincrease in chlorophyll content which may be due to the higheraccumulation of micronutrients such as Mn, Fe, Cu and Zn(Dwivedi et al., 2007).

3.3. Heavy metals contents in fly ash amended soil

The results of metal analysis showed an increment in the levelof DTPA extractable heavy metals in FA applied soil (Table 1) ascompared to control (FS). Up to FA10 no significant increment inthe concentrations of Zn, Fe, Cu, Mn, Cd and Cr were recorded inFA applied soils over the control. However, at FA20 there wassignificant increase in the concentration of all metals except Znand Cr over the control and initial soil. Phung et al. (1978) reportedthat application of highly alkaline FA to acidic soil decreased therelease of trace elements like Fe, Mn, Ni, Co and Pb whereas,release of these elements from alkaline soil remained unchanged.The FA used in our study had alkaline pH, hence increased level ofDTPA extractable heavy metals at higher dose of FA applicationcould be due to their high inherent concentration in FA asobserved by others (Lee et al., 2006; Adriano et al., 2002).

3.4. Heavy metals contents in rice grain and straw

Concentrations of metals in the rice grain of FA treated soil aresummarized in Table 2. Metals concentration in the rice grain andstraw were in the order Fe4Mn4Zn4Cu4Pb4Cr4Cd. Con-centration of heavy metals in grain and straw increased withincrease in the rate of FA application to soil. In general, across thetreatments the heavy metals content was higher in straw ascompared to grain except for Cu, which was higher in grain ascompared to straw. The observed differences in the metals con-centration in the grain and straw suggest different cellularmechanisms of bioaccumulation of metals that may control theirtranslocation in the plant systems. The availability, non-availabilityand bioaccumulation of metal depends on several environmental

Table 1DTPA extractable heavy metal concentration (mg kg-1) in coal fly ash amended soilafter the harvest of crop.

Treatments Zn Fe Cu Mn Pb Cd Cr

FS 0.75c 41.3d 0.78c 4.6c 0.80c 0.06c 0.16cFA5 0.74c 43.2d 0.81c 6.3c 0.82c 0.07c 0.18cFA10 0.75c 45.3d 0.85c 9.3c 0.89c 0.07c 0.18cFA20 0.78c 56.8c 1.01c 15.3bc 1.02bc 0.09bc 0.21cFA40 0.96b 69.6b 1.53b 21.6b 1.15b 0.11b 0.32bFA100 2.19a 89.3a 2.65a 35.7a 1.89a 0.28a 0.48a

Means with the same letter are not significantly (po0.05) different in a column.

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factors such as pH, solubility, soil mineralogy, texture, chemicalspeciation of the metal, presence of humic substances, otherorganic chelators, presence of other metals, and amorphous Feand Al content (Sinha et al., 2007). Mean comparison showed thatconcentrations of various heavy metals in grain and straw of ricewere significantly (po0.05) higher in 100 per cent fly ashamendments than the control. Increasing doses of fly ash havebeen reported to increase the heavy metals contents in the plants,with highest accumulations reported in the 100 per cent fly ashuse (Jala and Goyal, 2006; Pandey et al., 2009).

3.5. Microbial population

Soil microbiological properties including microbial biomass,microbial diversity, microbial activity and enzyme activity areimportant soil quality indicators (Nielsen and Winding, 2002)and often used to assess extent of soil pollution due to heavymetal contamination (Yang et al., 2006; Bloem and Breure, 2003;Rajapaksha et al., 2004). In this study, the application of FA up toFA40 did not result any change in aerobic heterotrophs and NH4

oxidizer population over the control (Fig. 1). The rhizosphere ofplants creates a more aerobic environment in soil that stimulatesmicrobial activity which enhances oxidation of organic chemicalresidues (Jones et al., 2004; Kirk et al., 2005). Population of bothfungi and actinomycetes decreased in FA amended soil as com-pared to the control, and beyond FA20 the growth of actinomy-cetes completely ceased (Fig. 1a). The fly ash used in this study wasalkaline in nature (pH 7.7), and many detrimental effects tomicroorganisms were attributed to high pH levels in the amendedsoils. There was slight decrease in NO2 oxidizer population in FAamended soil as compared to the control whereas denitrifiersshowed an increasing trend up to FA40 (Fig. 1b). It was earlierreported that fly ash amendment doubled the nitrification poten-tial of soils and resulted in higher numbers of denitrifiers in soils(Schutter and Fuhrmann, 2001). A significant reduction in popula-tion of all microorganisms was observed in FA100 (Fig. 1). Thisreduction in microbial population in soil at higher doses of fly ashis attributed to the lack of substrate C, inadequate N supply andhigh availability of heavy metals content in the fly ash (Table S1, inthe Supplementary materials section). Some authors haveobserved that the most limiting factors for microbial activity areusually a lack of substrate C as an energy source for heterotrophicmicroorganisms and the lack of an adequate N supply (Klubek etal., 1992). Surridge et al. (2009) have reported that fly ash additionhas a neutralizing effect on the soil leading to increased mobilityof nutrients, ultimately causing an increase in bacterial speciesrichness. However, fly ash also has a high content of toxicheavy metals (Jala and Goyal, 2006) which can hinder normalmicrobial metabolic processes when added in the soil at higherconcentrations.

3.6. Enzymatic activity

Soil enzyme activities are the direct expression of the soilcommunity to metabolic requirements and available nutrients(Caldwell, 2005). In general, enzyme activity is considered as agood index of soil quality because of their intimate relationship tosoil biology, ease of measurements, and rapid response to changein soil management practices (Dick et al., 1996). Activity of alkalinephoshpatase did not show any significant change when FA wasapplied at a rate of five per cent beyond which there was adecreasing trend, whereas there was significant reduction in acidphoshpatase activity even at lower dose of FA (Fig. 2a). FDAactivity was observed to increase with the application of FA froma value of 1.48 mg fluorescein g�1 soil in FS to a maximum of7.28 mg fluorescein g�1 soil in FA40; however, further increase ofFA to FA100 resulted in sharp fall in FDA activity to a value of1.57 mg fluorescein g�1 soil (Fig. 2b). Application of FA up to a levelof FA10 increased the activity of urease to a highest value of305.5 mg urea g�1 soil, beyond which there was gradual reductionin the activity and at FA100 it was at par with that recorded inFS (Fig. 2c). Some reports show that application of FA at the rate

Table 2Heavy metal concentration (mg kg-1) of rice grain and straw grown in coal fly ash amended soil.

Treatments Grain Straw

Zn Fe Cu Mn Pb Cd Cr Zn Fe Cu Mn Pb Cd Cr

FS 32.5bc 45.6c 6.4a 25.4b 0.42b 0.15b 0.22b 47.1d 56.5d 4.6bc 96.3bc 0.66d 0.53d 0.59dFA5 30.8c 44.8c 6.5a 25.7b 0.42b 0.15b 0.23b 45.2d 59.7cd 4.2d 88.4d 0.71cd 0.57d 0.68cFA10 31.9bc 45.9c 6.5a 25.5b 0.43b 0.14b 0.22b 48.7cd 61.2bcd 4.4cd 92.6cd 0.78bcd 0.59cd 0.74bcFA20 34.9b 49.6c 6.8a 26.9b 0.45ab 0.16b 0.24b 51.6bc 64.1abc 4.8b 97.3bc 0.84abc 0.67bc 0.77bFA40 35.3b 58.8b 6.7a 28.5b 0.46ab 0.19ab 0.28a 53.7ab 66.7a 4.9ab 101.5ab 0.87ab 0.72b 0.80bFA100 39.8a 68.5a 6.9a 33.5a 0.50a 0.22a 0.30a 56.5a 68.4a 5.2a 107.6a 0.98a 0.84a 0.97a

Means with the same letter are not significantly (po0.05) different in a column.

Fig. 1. Effect of different doses of coal fly ash on (a) aerobic heterotrophs (AH),actinomycetes (AM), fungi and (b) ammonium oxidizers (AO), denitrifiers andnitrite oxidizers (NO). Columns followed by common letters under each microbialgroup are not significantly (po0.05) different by Duncans' multiple range test(DMRT).

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of 10 t ha�1 was optimum for bacterial population, soil dehydro-genase activity and microbial biomass (Kohli and Goyal, 2010). FAadded at levels exceeding ten per cent, resulted in a decline inmicrobial activity (Kirk et al., 2005). Schutter and Fuhrmann(2001) observed that there was a trend for increased alkalinephosphatase activity in soils amended with fly ash. Sarangi et al.(2001) reported a significant increase in the rate of CO2 evolutionand the activities of soil enzymes with increasing application of FAup to 10 t ha�1, but decreased with further higher levels. Theenrichment of extractable fraction of heavy metals (Zn, Fe, Cu, Mn,Pb, Cd and Cr) along with other changes in soil conditions such aspH and labile carbon pools (data not presented) and reducedmicrobial population, created by fly ash at higher doses might bethe reason for inhibiting the enzyme activities in soil (Pati andSahu, 2004; Sarangi et al., 2001; Smejkalova et al., 2003). Decreasein different enzyme activities due to pH and various modes of toxicaction of different metals like Cd, Cr, Cu, Pb, Zn, etc., (antimeta-bolic, precipitation and chelation, decomposition of essentialmetabolites, decreasing cell membrane permeability and replace-ment of electrochemically important element in cells) has alsobeen reported by Giller et al. (1998) and Kao et al. (2006).

3.7. Microbial N transformation processes

Mineralization, nitrification and denitrification are importantsoil functions greatly affected by soil microbial activity and bothmineralization and nitrification rates are considered as sensitiveindicators to assess the response of soil to biological change(Stamatiadis et al., 1999). Studies on response of N transformationprocesses to FA application are very few and mostly limited tolaboratory incubation. We observed application of FA up to 40 percent did not show any significant change in the mineralization rate

over the control, however, there was a significant reduction in themineralization rate at FA100 (Table 3). Non-availability of carbonand very little content (traces) of N in the fly ash was attributed tothe no significant change in N mineralization for lower doses of itsapplication and for the reduced N mineralization in 100 per centFA treatment as compared to control. An increasing trend innitrification potential was observed in FA amended soil up toFA40 though there was no corresponding increase in population ofnitrifiers, whereas denitrifying enzyme activity showed a decreas-ing trend with the FA application. There are reports of reduction inN mineralization, nitrification and denitrification potential due tofly ash mixing (50 t ha�1) up to a depth of 40 cm in soil (Suwalka,2003). Laboratory studies also showed reduced nitrification poten-tial in soil amended with fly ash (Cervelli et al., 1987; Garau et al.,1991). Increasing trend in nitrification potential up to FA40observed in our study could be due to improved aeration of thesoil as the FA incorporation improves the physical condition of soil(Mishra et al., 2007).

4. Conclusion

At lower levels (up to twenty per cent), if applied to theagricultural soils, fly ash may enhance the micronutrient avail-ability and microbial activity and results in better crop growth,while higher dose of fly ash led to accumulation of toxic metals insoil and suppressed the microbial growth and activity. Applicationof fly ash up to 40 per cent in heavy texture soil does not interferein the N mineralization process due to enhanced aeration. How-ever, FA dose optimization need extensive field trials and will varybased on soil type and source of fly ash. Bioavailability ofaccumulated toxic metals and their entry into food chain shouldbe thoroughly studied. Besides, studies should be conducted toexamine the long term impact of FA application on soil quality.

Acknowledgments

The authors acknowledge the financial assistance througha research project by Department of Science and Technology,New Delhi.(Grant no. Fly ash 26.00.31).

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ecoenv.2014.03.033.

Fig. 2. Effect of different doses of coal fly ash on (a) phosphatase, (b) FDA activityand (c) urease activity of soil. Columns followed by common letters are notsignificantly (po0.05) different by Duncans' multiple range test (DMRT).

Table 3Microbial N cycling activity in coal fly ash-amended soil grown under rice crop.

Treatments Nitrogenmineralization(mg NH4–N kg�1

dry soil d�1)

Nitrification potential (mgNO3–N kg�1

dry soil d�1)

Denitrifyingenzyme activity(mg N2O kg�1

dry soil h�1)

FS 220ab 1.8c 16.1aFA5 200ab 2.3bc 16.2aFA10 240a 2.8b 15.4abFA20 240a 3.1b 14.1abFA40 240a 3.9a 12.5bFA100 180b 2.4bc 12.2b

Means with the same letter are not significantly (po0.05) different in a column.

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Please cite this article as: Nayak, A.K., et al., Effect of fly ash application on soil microbial response and heavy metal accumulation in soiland rice plant. Ecotoxicol. Environ. Saf. (2014), http://dx.doi.org/10.1016/j.ecoenv.2014.03.033i

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Please cite this article as: Nayak, A.K., et al., Effect of fly ash application on soil microbial response and heavy metal accumulation in soiland rice plant. Ecotoxicol. Environ. Saf. (2014), http://dx.doi.org/10.1016/j.ecoenv.2014.03.033i