Studies on the Efficiency of Eudrilus eugeniae in the Bioconversion of Tamarind Fruit Shell Waste Mixed with Diclofenac and Bisphenol-A

Earthworms are believed to have a relationship with soil microorganisms ranging from commensalism to species-specific mutualism (Sampedro and Whalen, 2007). Parle (1963) first reported the presence of microbes in the earthworm gut and several researchers followed and attempted to study the earthworm gut microbes using direct culture methods. Karsten et al., (1995) and Garg et al., (2006) have suggested that vermicomposting is a waste management technique that promotes the production of organic fertilizers from bioorganic wastes. Earthworms are the crucial drivers of the vermicompost process. They cause fragmentation of the ingested material through muscular action which increases the surface area for microbial activities (Edwards, 1988; Lazcano et al., 2008). In the earthworm gut a variety of intestinal microorganisms produce digestive enzymes such as amylase, proteases, lipases and celluloses enhance the biodegradation of organic matter (Aira et al., 2006).
       
The vermicomposts application is one of the effective methods to rejuvenate the depleted soil fertility and enrich the available pool of nutrients and conserve more water, maintain soil quality and conserve more biological resources. As reported by some of the researchers vermicomposting is an appropriate technology for residue and waste management (Jambhekar, 1992). Vermicomposting is an easy and effective way to recycle agricultural waste, city garbage and kitchen waste along with bioconversion of organic waste materials into nutritious compost by earthworm activity (Hemalatha, 2012). Vermicompost is potential organic manure rich in plant nutrients compared to farmyard manure or other organic manures in respect to supply of N, P and K fertilizers. The activities of dehydrogenase, nitrogenase, phosphates and urease have been found to higher during the process of vermicomposting. It is fast growing in popularity as a tool of reclamation of waste land (Bhawalkar, 1993, Arunkumar, 2000; Roy et al., 2000; Sinha et al., 2005).
       
Vermitechnology is an important aspect of biotechnology involving the use of earthworms for processing various types of organic wastes into valuable resources. Vermicomposting helps to process wastes simultaneously giving bio fertilizers and proteins (Prabha et al., 2005). Thus vermitechnology could successfully be used to clean the environment (Lal et al., 2003).
       
Soil nutrients are important contributors to soil fertility and humification processes. Of which, earthworms should be considered as a key-stone organisms in regulating nutrient cycling processes in many eco-systems (Tripathi and Bhardwaj, 2004). Earthworms can consume practically all kinds of organic wastes, consume two to five times its body weight and after using 5-10 per cent of the feed stock for its growth, excrete mucus coated undigested matter as nutrients and vitamins rich worm casts (Ismail et al., 2004). Vermicast is rich in mineral nutrients, vitamins, plant growth hormones, proteins and enzymes. Thus vermicast is considered as a very good organic fertilizer and soil conditioner. In the present study, the tamarind shell waste is used to convert waste into useful vermicast by using Eudrilus eugeniae and also assess the efficiency of the earthworm when the waste is mixed with environmental pollutants such as bisphenol-A and diclofenac.

Eudrilus eugeniae, the elusion earthworm species was collected from Mangalam, Thiruvanamalai district and Tamil Nadu. The collected earthworms were brought to laboratory for acclimatization. The study was carried out during the period of June 2019 to August 2019.
 
Experimental methods
 
In the experiment, pre-digested Tamarind shell waste was mixed with soil. The experimental set up was maintained in the laboratory with the introduction of earthworm Eudrilus eugeniae. In another pot, the same mixture was taken separately with bisphenol-A and Diclofenac and then released earthworm. The one fourth of the LC₅₀ value of diclofenac and bisphenol-A was mixed in the appropriate pot for this study (Bhuvaneswari, 2017 and Ravi, 2017).  Suitable control was maintained.
 
Control: Cow dung + Garden soil.
 
Experiment 1: Garden soil + Cow dung 25 gm + Tamarind shell waste 40 gm.
Experiment 2: Garden soil + Cow dung 25 gm + Tamarind shell waste 40 gm + Bisphenol-A.
Experiment 3: Garden soil + Cow dung 25 gm + Tamarind shell waste 40 gm + Diclofenac.
       
The waste mixtures were allowed to pass through earthworm guts for vermicomposting. The moisture content of the organic substrates in each pot was maintained between 60% and 65% throughout the study period by sprinkling water after every 24 hours. The experiment was conducted by randomized design with three replications. In the vermicompost materials the experimental set up was maintained till 30 days. After that, the compost material was analyzed for the physico-chemical, micro and macro nutrients such as pH, electrical conductivity, organic carbon, total nitrogen, total phosphorus, total potassium, total manganese, total zinc, total copper, total iron, total calcium, total magnesium and total sodium by the standard procedures. The statistical analysis was carried out to find out the significance of the results.

The conversion of Tamarind shell waste into vermicompost was observed by using Eudrilus eugeniae in our laboratory. The tamarind shell waste was mixed with bisphenol-A and diclofenac separately in the vermibed and seen the efficiency of the earthworm in the conversion of Tamarind shell waste into minerals. 
 
Results obtained from the tamarind shell waste mixed with bisphenol-A
 
(Table 1, 2 and Fig 1)
 

 

 

 
Mean number of days required for the biotransformation of waste into vermicompost was 30 days. The nutrients status of vermicompost depends on the type of waste material processed by the earthworm.
       
The cow dung used as the inoculants with bisphenol-A in the vermicomposting process showed breakdown of bio wastes resulting in the reduction of certain nutrients, such as the nitrogen, phosphorus, potassium, manganese, calcium and magnesium on 30^th day as compared to control. The total nitrogen, total phosphorus total potassium, total zinc, total copper, total iron, total manganese, total calcium, total magnesium and total sodium showed positive correlation with the other nutrients
       
The N value was decreased (0.869±0.02) in the bisphenol-A treated compost on 30^th day. The value was statistically significant. Similarly P was also decreased in the treatment where the level was 0.152±0.002 and showed significance at P<0.05. The analysis of Fe on 30^thday indicated decrease in the treatments. Statistical analysis revealed significance at P<0.01 level. Cu showed insignificant changes as compared to control. Manganese indicated significant decrease in the treatment on 30^th day.
       
Physico-chemical properties of the soil mixed with bispnenol-A in the Tamarind shell waste were analyzed on 30^th day along with control and experiment. There was a significant increase in the electrical conductivity (EC) in bisphenol-A treated Tamarind shell waste and the values in all the treatments were statistically significant at P<0.01 level. Similarly, pH value was decreased in all the treatments.
 
Results obtained from the Tamarind shell waste mixed with diclofenac
 
(Table 1, 3 and Fig 2)
 

 

 
The analysis showed the various levels of micro and macro nutrients. Among the nutrients, the important nutrient is the N which was increased significantly in the experiment (exp-1) and in the diclofenac treated experiment (expt-3) the analysis showed decreased the level of N than the control value. The nitrogen level of control was 1.216±0.002 whereas in the diclofenac treatment the level of nitrogen was decreased to 0.834±0.002. The study indicated that the optimum levels of mineralization takes nearly after 30 days. Similarly P in the expt-1 was increased and in the expt-3 the level was 0.166±0.001 and showed insignificance. The P level showed decrease by 0.166±0.001 when compared to experiment-1. In contrast to the N and P, the K level was decreased by 0.589±0.002 on 30^th day. Fe level was increased significantly in the experiment-1; however the analysis on 30^thday showed further decrease in the expt-2. In contrast to the decrease, Mg in the diclofenac treated waste revealed slight increase when compared to control. Manganese, calcium and sodium showed the decreased level. The statistical analysis also showed significance at P<0.01 level except Na level. Total nitrogen, total phosphorus, total potassium and total zinc revealed positive correlation with the pH in the correlation matrix.            
       
The electrical conductivity (EC) was increased in the expirement-1 and experiment-2. The pH value was slightly increased in the expt-1whereas in the experiment-2 it was decreased. The ‘t’ test showed insignificance in the Tamarind shell waste conversion and rest of the nutrients showed significance.
       
Soil biota plays an important role in supporting nutrient cycling as well as creating and stabilizing soil structure. Due to their dominant position in the soil they play a major role in the soil turnover and soil fertility. Earthworms are terrestrial invertebrates originated about 600 million years ago, during the pre-Cambrian era (Salmon et al., 2005) designated the earthworm  as major soil “ecosystem engineers” because of their physical, chemical and biological influence on soil.
       
In recent years, the problem of efficient disposal and management of organic solid wastes has become more vigorous due to rapidly increasing populations, intensive agriculture and industrialization. Production of large quantities of organic wastes all over the world poses major environmental and disposal problems. Vermicomposting is an eco-biotechnological process that transforms energy rich and complex organic substances into stabilized humuslike vermicompost. Benitez et al., (2005) reported that in vermicomposting process, inoculated earthworm maintains aerobic condition in the organic wastes. Vermicompost contain nutrients that are readily available for plant uptake, such as nitrates, exchangeable phosphorus and soluble potassium, calcium and magnesium.
       
The nitrogen content of the vermicompost of Tamarind shell waste was decreased on 30^th day and probably due to the mineralization of organic matter by earthworms during vermicomposting. The similarity between nitrogen content for the treatments with 10, 15 and 20 earthworms in cow, sheep and chicken wastes suggest that up to 10 individuals of Eudrilus eugeniae can be considered as the limit for earthworm density to prevent competition (Coulibaly et al., 2011; 2014).
               
Phosphorous content also in the bisphenol-A was decreased in this study. A significant effect of the number of earthworms on P content was observed only in the composting of pig waste at 60 and 90 day and of chicken waste at 90 day. At 60 day of composting, there was no difference in the P content in the pig waste obtained with 5, 10 and 15 earthworms. For pig waste, at 90 day of composting, the effect of the number of earthworms was variable while in chicken waste, the P values were similar in the treatments with 10 to 20 earthworms; all were higher than that of chicken waste treated with 5 earthworms (Edwards and Lofty 1997).
       
A decrease in P content during vermicomposting could have been induced by the mineralization and the mobilization of phosphorus through bacterial and faecal phosphatase activity of the earthworms. (Satchell and Martin, 1984; Le Bayon and Binet, 2006). The other nutrient K also showed decreased level bisphenol-A treatment. Similarly, Orozco et al., (1996) reported a decrease in K in the vermicomposting of coffee pulp waste. These differences could be attributed to the chemical nature of the initial raw wastes. The micro nutrients such as iron, manganese, zinc and copper showed heterogeneous levels on different days of analysis of vermicompost.
       
Vermicomposting is one of the methods through which organic manure and other agricultural organic waste can be recycled for maintenance of soil organic matter and for sustaining soil productivity (Edwards, 1998; Kale, 1998). Vermicomposting is an accelerated process of bio oxidation and stabilization of organic waste involving interactions between earthworms and microorganisms (Dominguez and Edwards, 2004).
       
The physical characters of the vermicompost obtained from Tamarind shell waste showed various levels on 30 day analysis. Similarly for cow waste there was no significant difference in the pH values with the use of different numbers of individuals of Eudrilus eugeniae at 30 day. At 60 day, the pH of the compost mineralized with 15 and 20 earthworms were similar, however, these values were lower than those of the composts decomposed with 5 and 10 earthworms. At the end of vermicomposting, the pH of the treatment with 5 earthworms was significantly higher than those of the treatments with 10, 15 and 20 earthworms. Regardless of the initial number of earthworms, the pH became acidic and was lower than the pH at 30 and 60 day (Coulibaly et al., 2011; 2014). The pH decreased when the number of earthworms increased during vermicomposting of sheep and chicken wastes.
       
A change in the electrical conductivity values in vermicompost may be due to the presence of exchangeable calcium, magnesium and potassium earthworm modifies substrate conditions, which consequently affects carbon losses from the substrates through microbial respiration in the form of CO₂ and even through mineralization of organic matter (Cambardella et al., 2003; Kaushik and Garg, 2003 and Garg et al., 2006). A large fraction of organic matter in the initial substrates was lost as CO₂ by the end of the vermicomposting period. The vermicomposted material had greater nitrogen content. The inoculation of worms in waste material considerably enhances the amount of N due to earthworm mediated nitrogen mineralization of wastes. It also suggested that the earthworm enhances the nitrogen levels of the substrate by adding its excretory products, mucus, body fluid, enzymes and even through the decaying tissues of dead worms in vermicomposting.

In this study, significant effects of bisphenol-A and diclofenac were observed in the vermicompost of tamarind shell waste. It may be due to the fact that digestive gland, a tissue involved in the control of metabolism is influenced by bisphenol-A and indirectly affected gene expression, including that of, antioxidant enzyme activities and lysosome functions. Overall, the results indicate that pollutants, at environmentally relevant concentrations, can have both distinct effects in invertebrates and modulating composting process.

The authors are thankful to the college principal and staff of Zoology Department for the permission and support in carry out the experiment.