Effect of Lignite Fly Ash on the Growth and Reproduction of Earthworm Eisenia fetida

Fly ash is an amorphous ferroalumino silicate, an important solid waste around thermal power plants. It creates problems leading to environmental degradation due to improper utilization or disposal. However, fly ash is a useful ameliorant that may improve the physical, chemical and biological properties of soils and is a source of readily available plant macro and micronutrients when it is used with biosolids. Supply of nutrients from fly ash with biosolids may enhance their agricultural use. The growth and reproduction of Eisenia fetida was studied during vermicomposting of fly ash with cowdung and pressmud in four different proportions (T1,T2,T3 & T4) and one control i.e., cow dung and pressmud alone. The growth, cocoon and hatchlings production were observed at the interval of 15 days over a period of 60 days. The maximum worm growth and reproduction was observed in bedding material alone. Next to that the T1 was observed as the best mixture for vermiculture.


Introduction
Fly ash, a resultant of combustion of coal at high temperature, has been regarded as a problematic solid waste all over the world.The conventional disposal method for fly ash leads to degradation of arable land and contamination of ground water 1 .The repeated exposure of fly ash causes irritation in eyes, skin, nose and results in arsenic poisoning 2 .
Coal combustion product generated each year in India is more than 100 mt per annum 3 of which 4 mt is released into the atmosphere.Coal combustion by-products were largely treated as waste materials.In fact, fly ash consists of practically all the elements present in soil except organic carbon and nitrogen.It was found that this material could be used as an additive/amendment material in agriculture applications.A careful assessment of soil and fly ash is required before its application as a soil-ameliorating agent 1 .The present outlets of fly ash disposal are using cement, concrete and grout industries 4 but such use only accounted for 38% of fly ash produced by thermal power stations 5 .The major problem faced by the coal/lignite thermal power stations all over the world is the handling and disposal of ash.In the thermal power station of the Neyveli Lignite Corporation Ltd., Neyveli, Tamil Nadu, India, ash pond spreads over an area of more than 45 ha and causes serious environmental hazards besides occupying fertile, cultivable land 6 .Mixtures of fly ash with organic waste have been tried by several authors [7][8][9] .
Menon 10 studied the effect of mixed application fly ash and organic compost on soil and availability and uptake of elements by various plant species.Very little is known regarding the effects of fly ash amendment on soil biological properties 11 .Use of fly ash as a soil-amending agent has been investigated for a variety of crops 12 .Fly ash acts as a potential dust insecticide against various pests infecting rice, vegetables etc.And also fly ash as a carrier in pesticide formulation and the role of fly ash in soil properties have been studied by several workers [13][14][15] .
The beneficial effect of earthworm on soil has been attributed to increase microbial populations and biologically active metabolites such as plant growth regulators 16 .Recycling of wastes through vermitechnology reduces the problems of non-utilization of agrowastes 17 .The quality and amount of food available influences the size of earthworm populations.The earthworms that are employed in organic wastes mixed with soil, to a certain extent accumulate toxic metals 18 and after vermicomposting they can be re-employed for the same purpose.
Earthworms growth, maturation, cocoon production and reproductive potential are not only influenced by environmental conditions but strongly affected by the quality and availability of food 19,20 .Growth and reproduction of different species of earthworms using different materials such as flax seeds 21 , cattle manure and goat manure 22 , pressmud 23 , leguminous leaf litter 24 have been studied.The decomposition efficiency of Perionyx sansibaricus for vermicomposting was evaluated by using a variety of wastes such as agriculture waste, farmyard manure and urban solid waste.However studies are scanty on the reproductive potential of earthworms influenced by fly ash 25 .
Vijayakumar and Narayanaswamy 26 applied 40 kg of fly ash/ha and reported 100% survival of earthworms.Bhattacharya and Chattopadhyay 27 found the survival of earthworms in the three different ratios (1:1, 1:3 and 3:1) of cowdung and fly ash.Ananthakrishnasamy 28 reported the survival of earthworms in three different ratios (1:1, 2:1 and 3:1) of cowdung and fly ash mixture.More recently the fly ash was vermicomposted with cowdung and the solubility of micro nutrients was analysed 29 .Apart from these, a study is available on the vermicomposting of fly ash with cowdung and reported about the variations in reproduction, zoomas and vermicast recovery 30 .So for, the studies available on the vermicomposting of fly ash are only with cowdung.
The present study was aimed to evaluate the effect of lignite fly ash on the growth and reproductive potential of E. fetida.During the period of 60 days the growth and reproduction were observed in fly ash (FA) with bedding material (BM -cowdung and pressmud) in various combinations where the pressmud is a solid waste produced by sugar mills.

Collection of earthworms
Eisenia fetida was obtained from the stock culture maintained in the Department of Zoology, Annamalai University.

Collection of fly ash (FA)
Fly ash (FA) was collected from the thermal power stations I, Neyveli Lignite Corporation, Neyveli, Tamil Nadu, India.

Collection of cowdung (CD)
The urine free cowdung was obtained from the experimental dairy farm in Annamalai University.The collected cowdung was sun dried and powdered, used as the substrate for the earthworm culture.

Collection of pressmud (PM)
One month old and cured pressmud (PM) was obtained from E.I.D Parry Sugar Mill at Nellikuppam, Cuddalore district, Tamil Nadu, India.

Experimental media and inoculation of worms
The cowdung and pressmud were mixed in the ratio of 1:1 (w/w) and used as bedding material (BM).Approximately 45 days old clittellate worms E. fetida (15 g/kg substrate) were inoculated in five different experimental media as mentioned below.The plastic troughs were covered with nylon mesh and maintained at the room temperature 27±2°C with 60-70% of moisture, the medium without fly ash were treated as control.In each combination six replicates were maintained.The experimental combinations were: -FA + BM (3:7) T 4  -FA + BM (4:6) T 5  -BM alone The biomass (wet weight) of worms was weighed in an electronic balance and the number of cocoons and hatchlings were counted by hand sorting method.The biomass (growth), cocoon and hatchling numbers were observed at the interval of 15 days, over a period of 60 days.The statistical significance of difference was tested using two-way ANOVA at 0.05 level.

Results and Discussion
The different levels of growth and reproduction of Eisenia fetida were observed in the fly ash + bedding material mixtures.Table 1-3 shows the values obtained for different parameters of growth and reproduction of E. fetida over the experimental period.

Growth and reproduction
The changes in worm biomass over the period of 60 days with various combinations of substrate are presented in Table 1.The percent changes in the biomass over the initial (0) day are given in brackets.Among the various experimental conditions such as T 1 , T 2 , T 3 , T 4 and T 5 , the maximum worm biomass was observed in 100% bedding material, i.e., in T 5 (25.6 ± 0.65 g), the least growth was observed in T 4 (18.3 ± 0.37 g) on 60 th day.
In all the experimental media the biomass have increased steadily but the biomass of E. fetida declined in T 3 on 15 th day and in T 4 on 15 th to 45 th day after inoculation of worms due to mortality.After 45 th day there was no mortality of worms and the biomass started to increase up to 60 th day (22% increases over the initial) due to decomposition of fly ash.The results obtained from all the treatments (T 1 , T 2 , T 3 , T 4 and T 5 ) clearly indicate that fly ash in combination with bedding material has positive effect on the growth of E. fetida.The efficiency of all the treatments supported the growth of E. fetida and it could be ranked in the following order T 5 (BM alone) > T 1 > T 2 > T 3 > T 4 .

T-Treatments Initial (0) -worm unworked substrates Mean ± SE of six observations (+/-) -percent change of increase /decrease over the initial
The inoculated worms started to produce cocoons and they were observed on 15 th day.On 60 th day among all the treatments the T 5 (68.5±1.04)shows best results when compared to the other treatments, it was followed by T 1 , T 2 , T 3 and T 4 respectively.The variation in the cocoon production is depicted in Table 2

T-Treatments Initial (0) -worm unworked substrates Mean ± SE of six observations
The hatchlings production was observed in all the treatments only on 30 th day.The greatest mean number of hatchlings per cocoon was observed in the 100% bedding material (T 5 ) followed by T 1 , T 2 , T 3 and T 4 (Table 3).The highest growth (biomass) and reproduction (cocoon and hatchlings) were observed in T 5 (BM alone) when compared to other treatments.The growth (biomass) and reproduction (cocoon production and hatchlings) of E. fetida were tested in the fly ash with bedding material.

T-Treatments Initial (0) -worm unworked substrates Mean ± SE of six observations
Our results showed that the maximum biomass, cocoon production and hatchlings were observed in bedding material alone (i.e.T 5 ), and it was followed by T 1 , T 2 and they were followed by T 3 and T 4 .
The potential of earthworms as waste processors has been well documented by various authors [31][32][33] .Nutrition is an essential factor to determine the maximum growth of an organism.The optimal growth, maturation, cocoon production and reproductive potential of earthworms have been reported to depend on the quality and quantity of the available feed and various physicochemical parameters 32,34 .The best results regarding nutritional quality of the vermicompost and the growth and reproduction of E. fetida were obtained when worms were allowed to feed on cowdung exclusively 35 .On the contrary it was reported that pressmud mixed with cowdung show precocious maturation of gonads in Lampito mauritii 23 .
Earthworms derive their nutrition from organic materials, living bacteria, fungi, diatoms, algae, protozoa, nematodes and decomposing animals.Neuhauser et al. 36 found that E. fetida gained weight, which was dependent on population density and food type.Growth and reproduction of P. excavatus, E. eugeniae in cattle dung were also reported by Reinecke et al. 37 .A nitrogen rich diet promotes rapid growth and increased cocoon production in P. excavatus 32 and a protein -rich substrate results in an increase in biomass and cocoon production in E. fetida 36 .
Greater percentage of solid textile mill sludge (STMS) in the feed mixture significantly affected the biomass gain and cocoon production 35,38 .Kaushik and Garg 38 results showed that the total number of cocoon production was maximum (62) in 100% CD and minimum (43) in 50% STMS feed mixture.Their results showed that CD amended with 20% and 30% STMS can be suitable growth medium for E. fetida.From the foregoing discussion it can be concluded that E. fetida is able to grow well in bedding material alone.At the same time T 1 BM feed mixture seems to be the better combination than the other combinations of fly ash.This could be probably due to contribution of more quantities of nutrients to the medium by the addition of more quantity of the bedding material.Hence fly ash can be utilized for vermiculture and vermicompost production in combination with bedding material.
In our observation, the maximum worm biomass was observed in T 5 (100% bedding material) compared to other treatments and the least growth was observed in T 4 .The efficiency of all the treatments to support the growth of E. fetida could be ranked in the following order T 5 > T 1 > T 2 > T 3 > T 4 .The T 5 showed superior growth of earthworms because the bedding material was having higher amount of nutrients.Compared to T 3 and T 4 , the combinations of T 1 and T 2 show better growth, it might be due to the higher percentage of bedding material which may provide higher nutrients for the growth of earthworms.

Conclusion
The outcome of the present investigation provides an alternative way for the utilization of fly ash apart from the conventional brick making, landfilling etc.The biomass increase and cocoon production of earthworm Eisenia fetida were high in 100% bedding material (cowdung and pressmud).The biomass and cocoon production were observed to have an inverse relationship with the percentage of fly ash mixed with bedding material.Our results support the findings of Ramalingam 32 , Kale 17 , Kaushik and Garg 38 , Garg and Kaushik 39 where they have mentioned the availability of food material influences the reproduction and growth of earthworm.The reduction in the biomass of earthworm up to 60 days in T 4 and up to 30 days in T 3 indicates that the increased percentage of fly ash reduces the growth of earthworms.

Table 1 .
Effect of fly ash -bedding material on the biomass of E. fetida (P<0.05)

Table 2 .
. Effect of fly ash -bedding material on the cocoon production (number) of E. fetida

Table 3 .
Effect of fly ash -bedding material on hatchability of E. fetida (P<0.05)