Earthworm and sugar industrial waste collection
In the present study young non-clitellated E. fetida were selected from a stock culture maintained in the vermicomposting unit of the Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, India. Cattle dung (CD) was arranged from the local dairy. Bagasse (B) was obtained from Rana Sugars Limited, Amritsar, India.
Experimental setup
Five proportions with different ratios of B and CD were prepared, namely, 0:100 (B0) 25:75 (B25), 50:50 (B50), 75:25 (B75) and 100:0 (B100) in plastic trays in triplicates were used for vermicomposting. The vermicomposting process was conducted for 135 days and almost 30 g of the substrate was collected on the first and last day of experiment as described earlier (Bhat et al. 2015a). The collected substrate from each tray was air dried, sieved and stored in polythene bags for genotoxicity analysis.
Allium cepa root chromosomal aberration assay
Extract preparation
The pre and post vermicompost samples were prepared according to the French Standard method (Ferrari et al. 1999) i.e. 1:10 (w:v) using double distilled water. The samples were shaken continuously for 24 h and filtered through Whatman filter paper No. 42 and the final extract was analyzed for root growth and genotoxicity studies. The extracts were subjected to 6 and 12 h treatment period to evaluate the frequency of chromosomal aberrations before and after vermicomposting.
Root growth test
Onion bulbs were placed on couplin jars containing different pre and post vermicompost extracts. The root length test was performed as a 96 h test (Rank 2003). The extracts were changed every after 48 h. After 96 h of experiment, the onion bulbs were washed in tap water and the best 10 root length of each onion was measured with the help of thread. The mean root length was calculated in centimeters.
Genotoxicity test
The genotoxicity of the pre and post vermicompost bagasse extracts was analysed using A. cepa root tip cells. The onions were denuded and were grown in coupling jars containing tap water for 24–36 h. The roots (0.5–1 cm) of onion bulbs were then placed in treatment jars containing different extracts of pre and post vermicompost bagasse (0, 25, 50, 75 and 100 %). The exposure time for each pre and post vermicompost extract was 6 and 12 h respectively. After the 3 and 6 h of treatment, the root tips were washed, fixed in farmer’s fluid (1:3, glacial acetic acid:ethanol) for 24 h and stored at cold temperature (4 °C).
Preparation of slides
The slides were prepared by hydrolyzing the root tips with 1 N HCl and then squashed in 9:1 ratio of aceto-orcein and 1 N HCl with intermittent heating for 1–2 min. After 25 min in aceto-orcein, the root tips were removed and immersed for 30 s in a drop of 45 % acetic acid. The root tips were then put on a clean slide, covered with a cover slip, squashed by match stick and sealed with a DPX solution. Each slide of the extract was labelled, examined/scored under microscope at magnification of 100× and photographs of normal and aberrant cells were taken. The total number of cells, diving cells and aberrant cells were counted in each pre and post vermicompost extract. Chromosomal abnormalities were scored in 450 cells of each extract. The chromosomal aberrations were classified into physiological (c-mitosis, delayed anaphases, laggard chromosomes, stickiness, vagrant chromosomes) and clastogenic (chromatin bridges and chromosomal breaks). The chromosomal aberrations were represented in percentage and were calculated by the number of aberrant cells as a percentage of total dividing cells for each extract. The cytotoxic activity of the pre and post vermicompost bagasse extracts was evaluated by the mitotic index (MI), through the number of dividing cells as the percentage of a total number of cells analyzed for each concentration.
$${\text{Mitotic}}\;{\text{index}} = \frac{{{\text{Number}}\;{\text{of}}\;{\text{dividing}}\;{\text{cell}}}}{{{\text{total}}\;{\text{number}}\;{\text{of}}\;{\text{cells}}}} \times 100$$
Statistical methods
The mitotic index and root length were presented as mean ± SE of triplicate experiment and the level of significance was determined by Student’s paired t test. The chromosomal aberrations were represented in percentage and the significance level was determined by Chi square test. Minitab version 14.0 was used for Statistical analysis.