Genotoxicity reduction in bagasse waste of sugar industry by earthworm technology
© The Author(s) 2016
Received: 20 April 2016
Accepted: 20 July 2016
Published: 27 July 2016
The aim of the present study was to assess the genotoxicity reduction in post vermicompost feed mixtures of bagasse (B) waste using earthworm Eisenia fetida. The genotoxicity of bagasse waste was determined by using Allium cepa root chromosomal aberration assay. Bagasse was amended with cattle dung in different proportions [0:100 (B0) 25:75 (B25), 50:50 (B50), 75:25 (B75) and 100:0 (B100)] on dry weight basis. Genotoxic effects of initial and post vermicompost bagasse extracts were analysed on the root tips cells of Allium cepa. Root length and mitotic index (MI) was found to be increased in post vermicompost extracts when compared to initial bagasse waste. The maximum percent increase of root length was observed in the B50 bagasse extract (96.60 %) and the maximum MI was observed in B100 mixture (14.20 ± 0.60) 6 h treatment which was similar to the control. Genotoxicity analysis of post vermicompost extracts of bagasse revealed a 21–44 % decline in the aberration frequencies and the maximum reduction was found in B75 extract (44.50 %). The increase in root length and mitotic index, as well as decrease in chromosomal aberrations indicates that E. fetida has the ability to reduce the genotoxicity of the bagasse waste.
KeywordsAllium cepa aberration assay Eisenia fetida Industrial waste Toxicity Vermicomposting
Enormous generation of industrial solid waste is a major environmental problem in the whole world. The improper disposal of these wastes can degrade the environment and affect the human health. The wastes generated from sugar industrial wastes are pressmud, bagasse, sugar beet mud and pulp (Bhat et al. 2014, 2015a, b). The management of these wastes are important in controlling the environmental pollution and contamination. Earthworm technology (vermitechnology) has the ability to reduce the toxicity of industrial wastes (Rezende et al. 2014). It involves degradation of organic waste into stable material by combined activities between earthworms and microbes living in their gut (Dominguez and Edwards 2011; Bhat et al. 2013; Haynes and Zhou 2016). Use of earthworms for toxicity reduction in industrial wastes has been used by many researchers (Jain et al. 2004; Srivastava et al. 2005; Bhat et al. 2014, 2015b). The chlorogocyte cells and the intestinal microflora of earthworms have the capability to decrease the genotoxicity of industrial wastes (Srivastava et al. 2005). Allium cepa root chromosomal aberration assay is widely used as a sensitive test in monitoring of waste genotoxicity (Rank 2003; Leme and Marin-Morales 2009). For monitoring environmental pollutants, this sensitive and stable test has also been adopted by the International Program on Plant Bioassay (IPPB; Ma 1999). The different endpoints of Allium cepa root chromosomal aberration assay used for assessment of genotoxicity in environment are mitotic index, chromosome aberrations, nuclear abnormalities and micronucleus (Leme and Marin-Morales 2009). Several researchers have used Allium cepa root chromosomal aberration assay in genotoxicity assessment of industrial wastes/effluents such as paint and textile industrial effluent (Samuel et al. 2010), electronic waste leachate (Bakare et al. 2012), tannery effluent (Masood and Malik 2013), pressmud sludge (Bhat et al. 2014), sugar beet mud (Bhat et al. 2015b), domestic sewage sludge (Mazzeo et al. 2015) etc. In the earlier study, Bhat et al. (2015a), bagasse waste amended with cattle dung was managed by vermicomposting process. The growth parameters of earthworm and physico-chemical analysis of feed mixture were done to know the nutrient content of vermicompost. In continuation of the previous study the vermicompost produced by the earthworm was assessed by genotoxicity test. In the current experiment A. cepa root chromosomal aberration assay was performed to evaluate the toxicity/genotoxicity reduction of the bagasse waste before and after vermistabilization.
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.
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
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.
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 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.
Results and discussion
Root growth test
Mean (±SE) root length of A. cepa exposed to initial and post vermicompost bagasse (B) extract for 96 h
Initial bagasse waste
Mean root length (cm)
Mean root length (cm)
3.66 ± 0.40
5.46 ± 0.41 (49.18)
3.54 ± 0.25
6.76 ± 0.36* (90.96)
3.24 ± 0.29
6.37 ± 0.21* (96.60)
2.96 ± 0.14
4.55 ± 0.31** (53.71)
2.18 ± 0.33
3.40 ± 0.17 (55.96)
Mitotic index and chromosomal aberrations
Effect of initial and post vermicompost extract of bagasse (B) on mitotix index of the root meristimatic cells of A. cepa
Mitotic index in initial bagasse wastea
Mitotic Index in post vermicompostb
0 % (control)
9.57 ± 0.34
11.83 ± 0.87
10.62 ± 0.41
12.88 ± 0.07*
9.97 ± 0.42
11.05 ± 0.42
10.74 ± 0.15
11.35 ± 0.22*
10.25 ± 0.53
12.27 ± 0.10*
11.53 ± 0.25
12.12 ± 0.34*
9.69 ± 0.42
12.45 ± 0.18**
9.07 ± 0.54
12.12 ± 0.51*
9.38 ± 0.03
14.20 ± 0.60*
10.17 ± 0.84
11.76 ± 0.58*
Chromosomal aberrations in the root tip cells of A. cepa exposed to initial and post vermicompost bagasse extracts
Types of chromosomal aberrations
0 % (control)
No. of Aberrant cellsa
No. of Aberrant cellsa
No. of Aberrant cellsa
No. of Aberrant cellsa
No. of Aberrant cellsa
Physiological aberrations (PA)
Clastogenic aberrations (CA)
Total aberrant cells (PA + CA)
Percent aberration (%)
Percent reduction (%)
Stickiness in the chromosomes may be due to DNA condensation or entanglement of chromatin fibers (Osterberg et al. 1984; Chauhan et al. 1999). Bridges in the chromosome may be due to the stickiness or by dicentric chromosome formation (Jabee et al. 2008). Breaks in chromosomes results from the fragile site breakage (Lukusa and Fryns 2008). Vagrant in chromosomes indicates spindle poisoning (Rank 2003). Bhat et al. (2014) have also observed that the percent aberration was higher (30.8 %) after initial exposure of pressmud sludge, but was reduced to 20.3 % after vermicomposting with E. fetida. The chlorogocyte cells and the intestinal microrganisms of E. fetida have the ability to detoxify the genotoxicity of industrial wastes (Jain et al. 2004; Srivastava et al. 2005). The present study suggests that initial feed mixtures of bagasse waste showed cytotoxic/genotoxic potential which declines at the end of vermicomposting. The results also revealed that the increase in root length and mitotic index, as well as decrease in chromosome aberrations in the post vermicompost extracts, indicates that E. fetida has the ability to detoxify the sugar industrial waste.
In the present study the results indicate the genotoxicity potential of bagasse waste and also the feasibility of earthworm technology to reduce the toxicity as observed by the results of A. cepa root chromosomal aberration assay. Increase in root length and mitotic index as well as decrease in chromosome aberrations in the post vermicompost proportions of bagasse waste indicates that the earthworm E. fetida has the ability to reduce the genotoxicity of this waste and the end product can be used safely in agriculture.
SAB: Performed the experiment and drafted the manuscript. JS: Provided guidance and improved the quality of the manuscript. APV: Formulated the objectives, provided resources and finalized the manuscript. All authors read and approved final manuscript.
Sartaj Ahmad Bhat is thankful to the UGC, New Delhi for the UGC-BSR Fellowship and Department of Botanical and Environmental Sciences Guru Nanak Dev University, Amritsar, for necessary research facilities.
The authors declare that they have no competing interests.
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- Bakare AA, Adeyemi AO, Adeyemi A, Alabi OA, Osibanjo O (2012) Cytogenotoxic effects of electronic waste leachate in Allium cepa. Caryologia 65:94–100View ArticleGoogle Scholar
- Bhat SA, Singh J, Vig AP (2013) Vermiremediation of dyeing sludge from textile mill with the help of exotic earthworm Eisenia fetida Savigny. Environ Sci Pollut Res 20:5975–5982View ArticleGoogle Scholar
- Bhat SA, Singh J, Vig AP (2014) Genotoxic assessment and optimization of pressmud with the help of exotic earthworm Eisenia fetida. Environ Sci Pollut Res 21:8112–8123View ArticleGoogle Scholar
- Bhat SA, Singh J, Vig AP (2015a) Potential utilization of bagasse as feed material for earthworm Eisenia fetida and production of vermicompost. Springerplus 4:11View ArticleGoogle Scholar
- Bhat SA, Singh J, Vig AP (2015b) Vermistabilization of sugar beet (Beta vulgaris L) waste produced from sugar factory using earthworm Eisenia fetida: genotoxic assessment by Allium cepa test. Environ Sci Pollut Res 22:11236–11254View ArticleGoogle Scholar
- Chauhan LKS, Saxena PN, Gupta SK (1999) Cytogenetic effects of cypermethrin and fenvalerate on the root meristem cells of Allium cepa. Environ Exp Bot 42:181–189View ArticleGoogle Scholar
- Dominguez J, Edwards CA (2011) Relationship between composting and vermicomposting. In: Edwards CA, Arancon NQ, Sherman R (eds) Vermiculture technology. CRC Press, Boca Raton, pp 11–25Google Scholar
- El-Ghamery AA, El-Nahas AI, Mansour MM (2000) The action of atrazine herbicideas an indicator of cell division on chromosomes and nucleic acid content in rootmeristems of Allium cepa and Vicia faba. Cytologia 65:277–287View ArticleGoogle Scholar
- Ferrari B, Radetski CM, Veber AM, Ferard JF (1999) Ecotoxicological assessment of solid waste: a combined liquid and solid phase testing approach using a battery of bioassays and biomarkers. Environ Toxicol Chem 18:1195–1202Google Scholar
- Grant WF (1999) Higher plant assays for the detection of chromosomal aberrations and gene mutations—a brief historical background on their use for screening and monitoring environmental chemicals. Mutat Res 426:107–112View ArticleGoogle Scholar
- Haynes RJ, Zhou YF (2016) Comparison of the chemical, physical and microbial properties of composts produced by conventional composting or vermicomposting using the same feedstocks. Environ Sci Pollut Res. doi:https://doi.org/10.1007/s11356-016-6197-0 Google Scholar
- Jabee F, Ansari MYK, Shahab D (2008) Studies on the effect of maleic hydrazide on root tip cells and pollen fertility in Trigonella foenum-graecum L. Turk J Biol 32:337–344Google Scholar
- Jain K, Singh J, Chauhan LKS, Murthy RC, Gupta SK (2004) Modulation of flyash-induced genotoxicity in Vicia faba by vermicomposting. Ecotoxicol Environ Saf 59:89–94View ArticleGoogle Scholar
- Leme DM, Marin-Morales MA (2009) Allium cepa test in environmental monitoring: a review on its application. Mutat Res 682:71–81View ArticleGoogle Scholar
- Lukusa T, Fryns JP (2008) Human chromosome fragility review. Biochim Biophys Acta 1779:3–16View ArticleGoogle Scholar
- Ma TH (1999) The international program on plant bioassays and the report of the follow-up study after the hands-on workshop in China. Mutat Res 426:103–106View ArticleGoogle Scholar
- Masood F, Malik A (2013) Mutagenicity and genotoxicity assessment of industrial wastewaters. Environ Sci Pollut Res 20:7386–7397View ArticleGoogle Scholar
- Mazzeo DEC, Fernandes TCC, Levy CE, Fontanetti CS, Marin-Morales MA (2015) Monitoring the natural attenuation of a sewage sludge toxicity using the Allium cepa test. Ecol Indic 56:60–69View ArticleGoogle Scholar
- Osterberg R, Persson D, Bjursell G (1984) The condensation of DNA by chromium (III) ions. J Biomol Struct Dyn 2:285–290View ArticleGoogle Scholar
- Rank J (2003) The method of Allium anaphase-telophase chromosome berration assay. Ekologija 1:38–42Google Scholar
- Rezende MOO, Dores-Silva PR, Silva MD, Zozolotto TCB, Landgraf MD (2014) Understanding the vermicompost process in sewage sludge: a Humic fraction study. Int J Agric For 4:94–99Google Scholar
- Samuel OB, Osuala FI, Odeigah PGC (2010) Cytogenotoxicity evaluation of two industrial effluents using Allium cepa assay. Afr J Environ Sci Technol 4:021–027Google Scholar
- Sharma S, Vig AP (2012) Genotoxicity of atrazine, avenoxan, diuron and quizalofop-P-ethyl herbicides using the Allium cepa root chromosomal aberration assay. Terr Aquat Environ Toxicol 6:90–95Google Scholar
- Srivastava R, Kumar D, Gupta SK (2005) Bioremediation of municipal sludge by vermitechnology and toxicity assessment by Allium cepa. Bioresour Technol 96:1867–1871View ArticleGoogle Scholar
- Sumitha KV, Thoppil JE (2015) Genotoxicity assessment of two common curing weeds: Hyptis suaveolens (L.) Poir. and Leucas indica (L.) R. Br. Cytotechnology. doi:https://doi.org/10.1007/s10616-015-9911-8 Google Scholar