¹Geobiotechnology Laboratory, National College (Autonomous), Tiruchirappalli – 620 001, Tamil Nadu, India.

²Post Graduate and Research Department of Biotechnology, National College (Autonomous), Tiruchirappalli – 620001, Tamil Nadu, India

³Post Graduate Department of Zoology, Khalsa College, Amritsar – 143 001, Punjab, India.

 ⁴Post Graduate and Research Department of Botany, National College (Autonomous), Tiruchirappalli – 620 001, Tamil Nadu, India.

Corresponding author email: senthil@nct.ac.in; microsenthilkumar@gmail.com

Article Publishing History

 INTRODUCTION

India is the second largest producer and exporter of textile and garments globally Corporate Catalyst (2015). Textile industries utilize about 200 L of water to produce 1 kg of fabric. Water is mainly used for application of chemicals onto the fibers and rinsing of the final products (Ghaly et al., 2014). The wastewater produced during this process contains large amount of dyes, salts, chemicals and heavy metals. Textile effluents are mostly discharged after minimal or no pretreatment into the neighboring water channels, streams and estuaries. The presence of chemicals in large quantities in the effluent not only affects water bodies but also collapses the soil properties adjacent to the water bodies and impinges on plant growth. Soil is a vibrant living matrix that is an indispensable part of the terrestrial ecosystem. The textile effluent polluted soil having an excess of any element or compound through direct or secondary exposure causes toxic response to biota or humans, resulting in unacceptable environmental risks (Baskaran et al.2009 Kabir et al. 2019,  Garczynska et al 2020).

The Textile belt of Tamil Nadu (Tirupur, Coimbatore, Erode and Salem), India, had been discharging their effluents into wastelands which were once cultivable until strict rules and regulations were passed to confine their effluents (Sadasivam et al. 2015). For the past two decades, the dyeing units located in the textile belt of Tamil Nadu have polluted non-perennial tributaries of Cauvery by discharging the toxic effluents. According to Tamil Nadu Pollution Control Board (TNPCB) 8.8 crore litres of effluents, after primary processing in effluent treatment plants are being let out into the Noyyal River every day. TNPCB specifies that the total dissolved solids (TDS) in the water discharged into the river should not be more than 2,100 parts per million (ppm). But the TDS level in the water in Orathupalayam dam area is above 9,000 ppm; in summer when evaporation shoot up, the level of TDS is still higher (Jayanth et al. 2011). Environmentally acceptable alternatives to unsustainable treatment methods for textile effluent polluted soils have been sought. Regulators are now starting to recognize the influence of contaminant bioavailability and mobility on environmental risk, and consequently, there is an increasing adoption of a risk-based approach when assessing soil quality ( Selvam et al. 2018 Garczynska et al 2020).

Organic materials are a popular choice for this as they are derived from biological matter and often require little pre-treatment before they may be directly applied to soils (Selvam et al. 2019). Additionally, such amendment practices may also be a convenient route to the disposal of organic residues surplus to requirement (Beesley et al. 2011). The amendments of textile effluent polluted soil for remediation has been a long standing procedure aimed at reducing the risk of pollutant transfer to proximal waters or receptor organisms ( Esmaeili 2020).

Hence, this study focuses on the biorestoration of textile effluent polluted soil of the Perundurai region, Erode, Tamil Nadu, India, through vermistabilization process. Cow dung was the organic material used in this study. Eudrilus eugeniae, commonly called African night crawler, is native of tropical West Africa, is an epigeic earthworm commonly used for vermistabilization as growth, fecundity, maturation and biomass are significantly greater when compared with other species. Hence, Eudrilus eugeniae was the choice for this study for vermi stabilization.

MATERIAL AND METHODS

Collection of textile effluent polluted soil (PS), cow dung (CD) and Eudrilus eugeniae

The PS was collected from the Common Effluent Treatment Plant (CETP) of State Industries Promotion Corporation of Tamil Nadu (SIPCOT) industrial estate area, Perundurai, Erode district, Tamil Nadu, India. Fresh cow dung was collected from the residential areas in and around Karumandapam, Tiruchirappalli, Tamil Nadu, India. The initial physico-chemical characteristics of PS and CD are given in Table 1. Eudrilus eugeniae were obtained from Periyar Research Organization for Bio-Technique and Eco-system (PROBE), Periyar Maniyammai University, Vallam, Thanjavur, Tamil Nadu, India.

Table 1. Initial physico-chemical characteristics of CD and PS

Composition	Cow dung (CD)	Polluted soil (PS)
pH	8.13±0.05	7.86±0.01
EC	1.63±0.05	0.62±0.01
TOC	56.30±0.45	4.26±0.01
TKN	12.33±0.05	7.64±0.04
TP	2.30±0.10	1.64±0.45
TK	9.33±0.05	9.40±0.17
C:N ratio	0.74±0.03	0.06±0.17
Cu	0.69±0.00	0.14±0.00
Zn	1.56±0.01	1.88±0.06
Fe	9.63±0.01	15.00±0.01
Cr	0.78±0.03	0.15±0.21

The experiments were conducted in a specially designed perforated plastic basket (diameter 20 cm and depth 15 cm) named as vermireactors and numbered as per PS and CD concentrations in each mixture. PS and CD were completely air-dried and sieved (2.0 mm mesh) before mixing. PS was mixed with CD in different ratios V_O 100% CD ,90:10, 80:20, 70:30,60:40, The study included a control vermireactor having CD only as feed mixture for the earthworms. The ratio of PS and CD in different ratio  These mixtures were turned manually every day for 15 days in order to eliminate volatile gases potentially toxic to earthworms. After 15 days, 3 adult individuals of E. eugeniae weighing between 1 g and 1.2 g were introduced into each vermireactor. The experimental period was for 8 consecutive weeks. Care was taken to rear the earthworms with more than 60 % of moisture in the immediate surroundings using water sprinkler. Moisture content was measured based on the previous studies of (Sadasivam et al. 2015). All the vermireactors were kept under ambient conditions (room temperature 25±2 °C; appropriate temperature for E. eugeniae). The experiments were repeated thrice for each feed mixture. Homogenized samples (free from earthworms, cocoons and hatchlings) of the feed material were drawn on 0, 7, 14, 21, 28, 35, 42, 49 and 56^th days from each vermireactors. The initial and final vermicompost samples were air-dried in the shade at room temperature, ground in a stainless steel blender and stored in plastic vials for further chemical analysis.

Physico-chemical characteristics and metal composition: The pH was measured using a digital pH meter (Systronics) by dissolving double distilled water suspension of each vermicompost in the ratio of 1:10 (w/v). Electrical conductivity (EC) was determined in double distilled water suspension of each mixture in the ratio of 1:10 (w/w) using a HM digital meter-COM-100. Total Organic Carbon (TOC) was measured on igniting the samples in a Muffle furnace at 550°C for 1 h following the method of (Nelson and Sommers (1996). Total Kjeldhal nitrogen (TKN) was determined by Bremner and Mulvaney (1982) procedure. Total phosphorous (TP) was analyzed using the colorimetric method of molybdenum in sulphuric acid. Total potassium (TK) was determined by flame photometer [Elico, CL 22D, Hyderabad, India]. Total sodium (TNa) was measured using a Systronics flame photometer-128 after digesting the samples in diacid mixtures (HClO₄: HNO₃ in 4:1 ratio). Heavy metals were measured by using Agilent AA 240 model atomic absorption spectrophotometer after digesting the samples in diacid mixtures.

Germination studies and plant growth measurements: Germination studies and plant growth measurements were done in Phaseolus radiatus (Green gram) to assess the manure quality of the biorestored soil. 1–5 % of the final vermicompost from the best vermireactor was mixed with normal soil to check germination and plant growth of  P. radiatus for 4 consecutive days.  Direct seed germination test (Warman 2010) of Phaseolus radiatus was conducted in triplicate by mixing final vermicompost of the best vermireactor with normal soil. Based on the previous studies carried out by David et al. (2014), the percentage amendment of vermicompost of the best vermireactor with normal soil chosen for this study ranged from 1 to 5 %. Amendments of the vermicompost of the best vermireactor with the normal soil were named as AST₁ to AST₅. Three controls were chosen (i) positive control comprising 100 % vermicompost of the best vermireactors (ASC₁), (ii) negative control comprising 100 % polluted soil (ASC₂) and (iii) overall control comprising 100 % normal soil (ASC₃). Each amended soil was seeded with 16 seeds of Phaseolus radiatus. The cell packs were moistened with potable water (as required) and kept under 12-14 h of light and 25±2°C in the area where vermicomposting had been carried out. Germination count of the seeds was noted after 48 hours of seeding. The plants were harvested after 4 days of sowing and repeatedly washed with tap water. Furthermore, these were rinsed with 10 mM CaCl₂ solution and washed with deionised water. Wet biomass of root, shoot and leaves was obtained after taking off excess water with tissue paper and using a Shimadzu weighing balance. Dry biomass of root, shoot and leaves was calculated by drying at 72° C in an oven for 72 h Wong et al. (1996)

Statistical analysis: All the samples were analyzed in triplicate (n = 3), and the results were averaged. Statistical analyses were performed by one-way analysis of variance (ANOVA) to evaluate the significant difference between vermireactors for growth and fecundity, physico-chemical and metal composition studies. Tukey’s post t-test was done to identify the homogenous types of vermireactors.  The number of seedlings and plants was counted and statistically analyzed using ANOVA (SPSS 16.0).

RESULTS AND DISCUSSION

The mean viability of worms was found to be highest in V₄ and lowest in V₇ of all the vermireactors, and mean viability was greatest at 2^nd week of the experimental period Fig. 1. Viability of E. eugeniae was zero in V₉ & V₁₀ from 1st to 8th week of the experimental period. The net mortality rate was found to be higher in V₇ & V₈ and consequently in V_c. Significant changes were observed in V₇ at 2^nd week, V₆, V₇ & V₈ at 3rd week, V₈ at 4^th week, V₃ at 5^th week and V₇ & V₈ at 8^th week when compared with all the other vermireactors in their respective week. The median viability of E. eugeniae was observed in V₁ and at 4^th week of the experimental period. The viability of the worms was found to be decreasing from 4^th week, and thereby, a gradual decrease in viability of worms was monitored till the end of the experiment. Mortality rate was directly proportional to increase in the concentration of polluted soil in the vermireactors. Increased concentration of PS in CD attributed mortality to degradation processes that result in changes in environmental characteristics (Garg and Kaushik. 2005). Addition of    at-least 50 % CD in PS was essential for the survival of E. eugeniae. Finally, these results suggested that feed mixtures in V₈, V₉ & V₁₀ cannot be used as substrate for vermicomposting by E. eugeniae, but must be supplemented with more CD. Similar observations had been reported by (Elvira et al 1998) for paper mill sludge vermicomposting by E. andrei and for textile mill sludge vermicomposting by E. foetida (Kaushik and Garg 2004).

Figure 1: All values are mean (n=3) S.D. Mean viability of the earthworms was compared among all the vermireactors having different concentration of CD+PS, where (a) highly significant and (b) less significant when compared with the control at (One Way ANOVA F ratio =0.988,TukeyTest

The mean worm weight of earthworm was found to be maximum in V₂ and lowest in V₁₀, and the highest mean worm weight was observed at 2^nd week of the experimental period Fig. 2. Significant changes were observed when V₇ & V₈ (except 0^th week of V₇ and 0th, 1st, 7th and 8^th week of V₈) were compared with rest of all the vermireactors from 1^st week to 8^th week of the experimental period. Increasing concentration of PS in vermireactors promoted a decrease in the weight of E. eugeniae, but did not show a regular pattern. The median worm weight was observed in V₄ when compared with all the other vermireactors and at 4^th week when compared with rest of all the weeks throughout the experimental period. Initial increase in worm weight (till 3^rd week in all the vermireactors except V₂ in which worm weight increased till 4^th week) was followed by stabilization. Later weight loss was observed in all the vermireactors from 4^th week except in V₂. The loss in worm weight can be attributed to the exhaustion of food (Renuka and Garg, 2007). When E. eugeniae received the food below a maintenance level, it lost weight at a rate which depended upon the quantity and nature of its ingestible substrates. This was in consonance with the study carried out by  Renuka et al. 2007  in E. foetida. (Garczynska et al 2020)

Figure 2: All values are insignificantly different (One Way ANOVA: =0.502, Tukey Test) where (a) highly significant and (b) significant when compared with the control

Cocoon production: The net cocoon production peaked in V_c and decreased in V₈ and V₆ in comparison with all the vermireactors; mean cocoon production increased at 6^th week and dropped at 8^th week of the experimental period Fig. 3. From 0^th to 8^th week, no cocoon production was observed in V₉ & V₁₀. However, least cocoon production was observed in V₈ from 3^rd to 7^th week. Significant alterations in cocoon production were observed when V₆ was compared with V₄, V₅ and V₇ at 2^nd week; V₈ with V₅, V₇ ,V₃ & V₄ at 3_rd week; V₆, V₇ V₈ with V₃, V4 &V₅ at 4_th week; V₆, V₇ &V₈ with rest of the vermireactors at 5^th week; V₆, V₇ &V₈ with V₃ at 6^th week; V₆, V₇ & V₈ with V₅, V₃& V₄ at 7^th week and finally when V₆ & V₇ were compared with the remaining vermireactors at 8^th week. The differences among rates of cocoon production in different vermireactors could be related to the biochemical quality of the feed mixtures, which is one of the important factors in determining the onset of cocoon production (Edwards et al.1998). The present study reported the maximum cocoon production in V₄ and at 5^th week during the entire treatment period. This is in consonance with the studies conducted by (Xie et al. 2012) which stated that addition of cow dung with sludge of animal wastewater treatment plant improved the mutual interaction between earthworm and microbes; the maximum proliferation of E. fetida was noted in sludge mixed with 40 % CD and proved to be a suitable growth medium for the fecundity of E. fetida.

Figure 3: All values are mean (n=3) S.D. Mean cocoon production was compared among all vermireactors having different concentration of CD + PS where (a) highly significant, (b) significant, (c) less significant, and (d) least significant when compared with the control at different letters in some column are significantly different (One Way ANOVA: F ratio 4.01, tukey test). V9 and V10 were not plotted in the graph since no cocoon production was observed throughout the experimental period.

Number of hatchling: The greatest mean number of hatchlings was produced in V_c and diminished in V₈. This observation was same as in the case of cocoon production. Dissimilarity in mean number of hatchlings was noted when maximum number of hatchlings peaked at 7^th week for all the vermireactors Fig. 4.  No significant changes were observed at 2^nd week of the treatment period. However, significant variations were examined when V₄, V₆ &V₈ were compared with all the other vermireactors at 3^rd week; V₆ &V₈ with V₄, V₅ & V₇ at 4^th week; V₆, V₇ &V₈ compared with V₄ &V₅ at 5^th week; V₆, V₇ & V₈ with V₃, V₄ & V₅ at 6^th and 7 ^th week and V₆, V₇ & V₈ with rest of all the vermireactors at 8^th week. The median cocoon production was observed in V₄ and at 5^th week among the entire treatment period. These data suggested that greater percentage of PS in feed mixtures significantly affected the development of hatchlings per cocoon. The feed, which provide earthworms with sufficient amount of easily metabolizable organic matter and non-assimilated carbohydrates, favors the growth and reproduction of the earthworms (Garg and Kaushik. 2005). The maximum number of hatchlings in V₄ was supported by the studies conducted by (Bhat et al. 2015) where maximum number of hatchlings was observed in 60-100% amendment of cow dung with sugar beet mud using E. fetida.

Figure 4: All values are mean (n=3)  S.D. Mean hatchling production was compared among all vermireactors having different concentration of CD + PS where (a) highly significant, (b) significant, (c) less significant, and (d) least significant when compared with the control at different letters in some column are significantly different (One Way ANOVA) : P < 0.05, tukey test).

Physico-chemical properties:  The physico-chemical characteristics of initial and final feed mixtures of textile effluent are shown in Table 2.Significant difference in pH was not observed in all the vermireactors of the initial feed mixtures. The pH of the initial feed mixtures ranged from 7.52 to 8.89, near neutral to alkaline. But the pH of the final vermicompost was near neutral in all the vermireactors except V₉ & V₁₀. The optimum pH was observed in V₃ & V₆ in the final vermicompost. It has been reported (Ndegwa and Thompson, 2000) that the pH shift is dynamic and substrate dependent. The shift in pH during vermicomposting process could be attributed to the production of metabolic compounds of aerobic digestion of organic stuffs such as CO₂, ammonia, NO₃^– and organic acids (Lopez et al., 2002).

Significant decrease in EC was observed in the final vermicompost except in V₉ & V₁₀ when compared with the initial feed mixtures of all the vermireactors. The net EC loss was 42 % in the final vermicompost when compared with the initial feed mixtures. The optimum EC was observed in V₄ & V₅ of the final vermicompost. Decrease in EC of the final vermicompost may be due to stabilization of mixtures (Singh et al. 2010). They also observed a decline in EC in the vermicompost from the biosludge of a beverage industry.

Total Organic Carbon (TOC): TOC of the final vermicompost was significantly reduced when :compared with the initial feed mixtures. The optimum TOC was observed in V₅ of the final vermicompost. There was a net loss of 52 % TOC in the vermireactors at the end of vermicomposting period. This finding was supported by (Kaviraj and Sharma, 2003) who reported 45% loss of carbon during vermicomposting of municipality and industrial wastes. (Suthar, 2006) reported that earthworms promoted microclimatic conditions in the vermireactors that increased the loss of TOC from substrate through microbial respiration (Abbas Esmaeili et al 2020)

Total Kjeldhal Nitrogen (TKN): A significant increase in TKN was recorded in all the vermireactors at the end of the study period. The net TKN of the final vermicompost increased upto 50 % and the nitrogen enhancement rate was optimum in V₃. Body secretions of earthworm (excreta, mucus) add nitrogen in substrate if earthworms were inoculated in organic wastes for longer periods. Earthworms also alter the microclimatic conditions of vermireactors which consequently promote microbial populations responsible for nitrogen enrichment (Suthar et al. 2012). Hence in this study, the combination of the organic waste (CD) and polluted soil acted as good energy stuff for nitrogen fixing bacteria enhanced by E. eugeniae.

Carbon Nitrogen ratio (C: N ratio):C: N ratio was found to decrease significantly (75 % net loss) in all the vermireactors of the final vermicompost when compared with initial feed mixtures. The optimal C:N ratio was observed in V₅ & V₆ of the final vermicompost. Decline in C:N ratio in this study was due to higher loss of carbon through microbial respiration in the form of CO₂ along with an increase in nitrogen and stabilization of waste by the action of E. eugeniae. Similar results were observed by (Bhat et al. 2014). Earlier studies have suggested that a C:N ratio below 20 is an indicative of acceptable maturity, while a ratio of 15 or lower is being preferred for agronomic use of composts. The vermicomposts obtained in this study showed C:N ratio within the preferable limits as described by (Morais and Queda, 2003).

Total Phosphorous (TP): Trivial changes were observed in phosphorous content in all the vermireactors of initial feed mixtures. The net P content in vermicomposted material was 118 % higher than in the initial feed mixtures. The optimal P content was observed in V₃ of the final vermicompost. Significant changes in phosphorous content were observed in all the vermireactors of the final vermicompost except in V₉ & V₁₀. The concentration of phosphorous in vermicomposted material may reflect the amount of organic forms of phosphorous in PS, but its mineralization rate is directly affected by the nature of the amendment material and activities of P-mineralizing microflora in decomposting wastes. (Satchell and Martin 1984) found an increase of 25 % in total P of paper-waste sludge after vermicomposting. In the present study there was 118 % increase in P content. This increase might be due to the release of P in available forms which were partly performed from earthworm gut phosphatases, and further release of P might be attributed to the P-solubilizing microorganisms present in worm casts as suggested by (Suthar et al. 2012). The results clearly suggest that P enrichment is directly related to the quality of material used in the vermireactors.

Total Potassium (TK): High potassium content was observed in the initial feed mixtures, but did not show a regular pattern and started diminishing after V₇. Subsequently, significant increase of mean TK content (43%) was observed in the final vermicompost when compared with initial feed mixtures and started diminishing after V₈. The optimal K content was observed in V₅ & V₈. Most of the earlier works on vermicomposting ( Bhargava 2009). ( Hait and Tare 2011) had reported higher K levels at the end of vermicomposting. The increase in K content indicates that when organic waste passes through the gut of earthworm, some fraction of organic materials is converted into more available types  of nutrients (i.e. exchangeable forms) due to the action of endogenic and/or exogenic enzymes Suthar ( 2010).

Heavy metal composition: Copper and Zinc (Cu & Zn):Transition metals Cu and Zn increased significantly (P<0.05) over initial feed mixtures in all the vermireactors except V₁₀.  Table 2. The mean increase in Cu and Zn content was 148% and 23%, respectively, in the final vermicompost. The optimal Cu and Zn content was observed in V₄ and V₅, respectively. In the final vermicompost, a little increase in Cu and Zn was noticed, and their contents increased with the increasing proportion of PS in the vermireactors. Increase in Cu and Zn metal contents may be due to decline in the weight and volume of the feed mixtures as suggested by Deolalikar et al. 2005. An increase in the content of transition metals in vermicompost from industrial sludge was also observed by Singh et al. 2010.

Table 2. Initial and final physico-chemical parameters (mean ± SE) different concentration of textile effluent polluted soil with cow dung

Parameters		VC	V1	V2	V3	V4
pH	Initial	8.13±0.05	7.77±0.11	7.73±0.12	7.76±0.12	7.63±0.47
	Final	7.86±0.00	7.43±0.02	7.27±0.02	7.58±0.05	7.59±0.05
	% change	-5.33	-4.37	-5.92	-2.37	-0.52
EC (Ds/m)	Initial	1.63±0.05	1.40±0.02	1.30±0.00	1.35±0.51	1.29±0.00
	Final	0.88±0.00	0.56±0.07	0.87±0.00	0.92±0.00	0.80±0.05
	% change	-46.01	-60	-21.18	-20.10	-42.66
TOC (k/kg)	Initial	81.30±0.04	63.33±0.57	64.46±0.55	66.06±0.94	61.30±0.10
	Final	25.63±0.36	25.50±0.55	26.63±0.45	27.20±0.17	28.46±0.11
	% change	-68.17	-57.73	-58.60	-58.82	-53.67
TKN (k/kg)	Initial	12.33±0.05	10.13±0.57	11.43±0.57	11.23±0.57	10.83±0.63
	Final	19.66±0.09	15.64±0.45	16.33±0.39	16.57±0.39	17.46±0.11
	% change	59.44	54.33	42.86	47.55	61.21
C:N ratio	Initial	6.32±0.29	6.25±0.37	5.72±0.40	5.96±0.11	5.34±0.21
	Final	1.36±0.05	1.70±0.03	1.63±0.03	1.62±0.04	1.63±0.14
	% change	-78.48	-72.8	-71.50	-72.81	-69.47
TP (g/kg)	Initial	2.30±0.10	1.90±0.17	1.76±0.57	1.63±0.45	1.60±0.10
	Final	4.57±0.32	3.56±0.11	3.70±0.34	3.56±0.11	4.64±0.45
	% change	98.69	87.36	53.40	110.22	89.29
TK (g/kg)	Initial	9.33±0.05	13.53±0.20	13.56±0.30	13.40±0.26	13.03±0.11
	Final	15.64±0.36	18.66±0.06	17.64±0.45	18.26±0.11	18.73±0.57
	% change	67.63	37.91	52.95	36.26	43.74
Cu (mg/kg)	Initial	0.69±0.00	0.35±0.06	0.30±0.06	0.23±0.32	0.25±0.12
	Final	0.72±0.01	0.79±0.00	0.91±0.01	0.96±0.01	0.82±0.01
	% change	4.35	55.69	67.03	317.12	227.99
Zn (mg/kg)	Initial	1.56±0.01	1.57±0.12	1.71±0.04	1.57±0.06	2.01±0.07
	Final	2.36±0.00	1.77±0.19	1.85±0.11	1.80±0.19	2.08±0.08
	% change	51.28	13.47	8.18	14.67	3.48
Fe (mg/kg)	Initial	9.63±0.01	16.44±0.02	14.36±0.18	15.33±0.00	15.20±0.00
	Final	9.12±0.00	12.68±0.11	12.89±0.00	11.69±0.00	12.58±0.01
	% change	-5.29	-22.87	-10.23	-23.74	-17.23
Cr (mg/kg)	Initial	0.12±0.01	0.39±0.00	0.48±0.01	0.53±0.04	0.47±0.01
	Final	0.05±0.00	0.04±0.01	0.05±0.00	0.02±0.00	0.06±0.11
	% change	-58.33	-89.74	-89.58	-96.22	-87.23

Significance level was determined by Tukey test p ≤ 0.05 Weight in (g/kg) and (mg/kg)

Parameters		V5	V6	V7	V8	V9	V10
pH	Initial	7.62±0.49	7.66±0.10	7.62±0.42	7.52±0.82	8.02±0.01	8.89±0.01
	Final	7.59±0.05	7.58±0.55	7.53±0.05	7.46±0.05	7.94±0.41	7.89±0.01
	% change	-0.39	-1.04	-1.18	-0.79	0.99	-12
EC (Ds/m)	Initial	1.10±0.07	1.17±0.05	1.10±0.05	1.10±0.01	0.59±0.00	0.62±0.05
	Final	0.92±0.00	0.57±0.05	0.87±0.00	0.78±0.00	0.49±0.00	0.32±0.05
	% change	-38.66	-52.99	-20.90	-29.09	-16.94	-48.38
TOC (k/kg)	Initial	61.10±0.56	28.96±0.56	21.43±0.15	19.16±0.20	6.41±0.30	5.26±0.12
	Final	25.64±0.45	22.23±0.05	12.33±0.57	10.64±0.45	5.63±0.05	4.26±0.11
	% change	-59.67	-23.18	-42.46	-44.62	-12.16	-19.01
TKN (k/kg)	Initial	9.73±0.57	10.80±0.60	9.43±0.57	10.76±0.57	8.64±0.45	7.64±0.45
	Final	17.23±0.05	16.83±0.05	16.16±0.05	15.54±0.05	9.24±0.45	8.64±0.45
	% change	77.08	55.83	71.36	44.42	6.94	13.08
C:N ratio	Initial	4.27±0.46	2.43±0.21	2.27±0.14	1.78±0.08	1.10±0.04	1.00±0.02
	Final	1.49±0.04	1.52±0.34	1.27±0.18	1.47±0.16	1.90±0.00	1.89±0.01
	% change	-76.23	37.44	-44.05	-17.41	-72.77	-88.99
TP (g/kg)	Initial	2.53±0.15	1.76±0.49	1.40±0.16	1.40±0.17	1.31±0.15	1.20±0.14
	Final	4.73±0.05	3.73±0.11	2.16±0.45	2.26±0.11	1.90±0.15	1.25±0.14
	% change	86.95	111.93	54.28	61.42	45.30	4.16
TK (g/kg)	Initial	12.43±0.11	12.56±0.64	11.13±0.11	11.10±0.10	11.11±0.90	10.10±0.90
	Final	18.43±0.57	19.26±0.11	19.26±0.11	18.64±0.45	13.17±0.90	11.85±0.64
	% change	48.23	53.34	73.04	67.92	18.54	17.32
Cu (mg/kg)	Initial	0.29±0.30	0.54±0.26	0.42±0.12	0.35±0.60	0.24±0.60	0.14±0.00
	Final	0.84±0.00	0.92±0.00	0.76±0.01	0.62±0.04	0.54±0.04	0.16±0.64
	% change	189.65	70.37	80.95	77.14	125.00	14.28
Zn (mg/kg)	Initial	1.84±0.13	1.50±0.17	1.79±0.71	1.85±0.84	1.90±0.84	1.88±0.06
	Final	2.25±0.95	2.36±0.11	2.39±0.49	2.45±0.00	2.32±0.00	1.99±0.00
	% change	22.28	57.33	33.51	32.42	22.10	5.85
Fe (mg/kg)	Initial	16.18±0.05	18.24±0.10	14.58±0.04	15.58±0.01	15.01±0.01	15.00±0.01
	Final	11.56±0.11	11.36±0.00	12.65±0.00	13.45±0.00	13.45±0.00	14.11±0.10
	% change	-28.55	-37.71	-13.23	-13.67	-10.39	-5.99
Cr (mg/kg)	Initial	0.42±0.00	0.43±0.04	0.52±0.00	0.61±0.02	0.13±0.00	0.16±0.00
	Final	0.04±0.01	0.05±0.01	0.03±0.01	0.02±0.00	0.05±0.05	0.12±0.21
	% change	-90.47	-88.37	-94.23	-96.77	-61.53	-25.00

Significance level was determined by Tukey test p ≤ 0.05 Weight in (g/kg) and (mg/kg)

Iron, and Chromium (Fe, and Cr): Significant decrease of Fe and Cr content was observed within and between initial feed mixtures and final vermicompost of all the vermireactors.  Table 2. The net decrease in Fe,  and Cr content in the final vermicompost was 20 %, 20 % and 86 %, respectively. The optimal Fe content was observed in V₄ and optimal . Heavy metal content of Fe, and Cr in this study was found to be decreased in the final vermicompost when compared with initial feed mixtures. Similar observations were made in the studies conducted by Garg and Kaushik, (2005) on vermistabilization of textile mill sludge and by Bhat et al. (2015) on vermistabilization of sugar beet by E. foetida. The decrease in the heavy metal content of Fe,  and Cr in the present study suggested that the vermicompost can be used in the fields without any ill effects on soil.

Germination studies and plant growth measurements: Based on the optimal characteristics of almost all the physico-chemical parameters, metal composition, growth and fecundity studies, the final vermicompost of V_5­ was chosen for amendment with normal soil in germination and plant growth measurements. Seed germination and plant growth were observed in all the amended soils for except the 100% polluted soil (ASC₂).  Charring of seeds was observed in ASC₂. 100 % of the final vermicompost of V_5­ (ASC₂) showed germination of the seeds, but plant growth was absent Table 3. Significant changes were observed when ASC₁ and ASC₂ were compared with ASC₃ and the rest of the treated amended soil (AST₁–AST₅). As the concentration of biorestored soil in the amended soil increased, plant growth of P. radiatus also increased except in the case of AST₅. Vegetative growth parameters of P. radiatus (root, shoot and leaf length) at all tested concentrations of amended soil were similar to normal soil. The maximum root and shoot length were observed in AST₃ and maximum leaf length in AST₄. Maximum wet biomass of root, shoot and leaves was observed in AST₁-AST₃, AST₄ and all the amended soils except ASC₃, respectively. Maximum dry biomass of root, shoot and leaves was observed in AST₄, AST₁ and all amended soils except ASC₃, respectively. The maximum protein content for root was observed in AST₄, for shoot in AST₅ and for leaves in AST₃ Table 3. Similar seed germination and vegetative growth were observed in Phaseolus mungo when 10% distillery sludge was amended with garden soil by Chandra et al. 2008.

Table 3. Effect of different concentration of bio-restored soil from V5 with normal soil on root, shoot, and leaves length and their respective biomass of Phaseolus radiatus

Treatment	No. of grown plants after 4th day	Root length (cm)	Wet biomass of root (g/plant)	Dry biomass of root (g/plant)	Shoot length (cm)	Wet biomass of shoot (g/plant)	Dry biomass of shoot (g/plant)	Length of leaves (cm)	Wet biomass of leaves (g/plant)	Dry biomass of leaves (g/plant)
ASC3	11.66 ± 0.57	9.55 ± 0.32	0.020	0.0050	10.39 ± 0.23	0.200	0.021	1.86 ± 0.23	0.027	0.0079
AST1	11.66 ± 0.57	10.23 ± 0.94	0.267	0.0061	12.36 ± 1.34	0.247	0.022	2.03 ± 0.15	0.054	0.0088
AST2	12.66 ± 0.57	10.90 ± 1.52	0.267	0.0056	12.50 ± 1.89	0.220	0.021	1.93 ± 0.11	0.054	0.0088
AST3	12.66 ± 1.15	9.91 ± 0.43	0.267	0.0058	9.86 ± 0.74	0.240	0.023	2.56 ± 0.20	0.054	0.0088
AST4	13.20 ± 2.64	9.86 ± 0.74	0.040	0.0062	11.95 ± 0.80	0.250	0.021	2.46 ± 0.32	0.054	0.0091
AST5	11.33 ± 0.57	9.86 ± 0.74	0.053	0.0059	11.95 ± 0.80	0.240	0.021	1.86 ± 0.20	0.054	0.0088

All values are mean (n = 3) ±S.D.

ASC3 : Amended Soil Control 3 (Overall control: 100% garden soil) AST: Amended Soil Treated

AST1 : 198 : 2 g (Garden soil : g)

AST2 : 196 : 4 g

AST3 :194 : 6 g

AST4 : 192 : 8 g

AST5 : 190 : 10 g

CONCLUSION

Restoration of textile effluent polluted soil by environmentally acceptable means has become a great challenge and a major concern of the present decade. This study had demonstrated vermistabilization by E. eugeniae as an appropriate technology for management of textile effluent polluted soil. Growth and fecundity studies of E. eugeniae revealed that a high degree of PS stabilization was achieved in (V₄ and V₅) (CD₄₀ PS₆₀ and CD₅₀ PC₅₀) at 4^th and/or 5^th week of the study period. The optimal CD+PS composition for almost all the favorable Physico-chemical parameters and metal concentrations ranged from (V₃ to V₆ ) (CD₇₀ PS₃₀ to CD₄₀ PS₆₀) when compared with V_c (CD₁₀₀  PS₀ )  (V₉ & V₁₀ ) (CD₉₀ PC₁₀) and (PC ₁₀₀), while (V₁ & V₂) (CD₉₀, PS₁₀ CD₂₀ and PC₈₀) on the other hand V_c ,(CD₁₀₀),  (V₉ and V₁₀) (CD₁₀ PC₉₀ and CD₀ PS₁₀₀) were not found to be suitable for the growth of E. eugeniae. Germination studies and plant growth measurements of Phaseolus radiatus showed that 1-5% amendment of the vermicompost of V₅ would serve as a soil conditioner or manure in the agricultural fields. The study revealed the fact that PS could be restored and transformed into good quality manure by vermicomposting when mixed with at least 30-50 % CD.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the TDT Division, Department of Science and Technology (DST), New Delhi, India [Grant No:DST/TDT/WMT/2017/054] and Tamil Nadu State Council for Science and Technology (TNSCST), Tamil Nadu, India [Grant No. TNSCST/S&T projects/VR/ES/2012-2013-200 dated 10.05.2013] for the financial support. The authors are grateful to the Dr. K. Anbarasu, Director of Studies and Shri. K. Ragunathan, the Secretary of National College (Autonomous), Tiruchirappalli, India, for all their constant support and encouragement in the pursuit of this research. The authors are indebted to Mr. K. Raja, Assistant Professor, Department of Mathematics; Dr. M. Murali, Assistant Professor, and Ms. V. Sathya, Assistant Professor, Department of Chemistry, National College (Autonomous), Tiruchirappalli, Tamil Nadu, India, for their help in the use of ORIGIN 8 software.

Conflict of interest: The authors declare that they have no conflict of interest.

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