Histological anomalies and alterations in enzyme activities of the earthworm Glyphidrillus tuberosus exposed to high concentrations of phosphogypsum

  • Soumya Nayak
  • C. S. K. Mishra
  • B. C. Guru
  • Suryasikha SamalEmail author


Phosphogypsum (PG) is the major solid waste generated by phosphate fertilizer plants and is used worldwide as sulfur and calcium supplement in agricultural soil. Considering the probability of elevated doses of PG during agricultural applications, this study was carried out to assess its impact on the connective tissue, tissue cholinesterase (ChE) activity, lactate dehydrogenase (LDH) activity, and lipid peroxidation (LPX) level of the tropical earthworm Glyphidrillus tuberosus (Stephenson) found in abundance in the rice fields in India. Consistent loss of connective tissue and sloughing of the intestinal epithelium were observed in worms exposed to 10%, 15%, and 20% concentrations of PG in soil over an incubation period of 30 days. ChE, LDH activities, and the level of LPX indicated highly significant variation (p < 0.01) between pre and postclitellar regions of the worm and concentrations of treatment. ChE activity was higher in postclitellar with respect to preclitellar region; however, the values for LDH activity and LPX level were higher in preclitellar region in comparison to postclitellar region in both PG treated and control worms. It was concluded that PG concentration at and beyond 10% could cause damage to muscle fibers and bring about significant alterations in these enzyme activities in G.tuberosus thus affecting the physiology and ecological functions of these worms.


Glyphidrillus tuberosus Phosphogypsum Connective tissue Lipid peroxidation Lactate dehydrogenase Cholinesterase 



The authors are thankful to Prof. R.K. Das, Head of the Department of Anatomy and Histology, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture and Technology, Bhubaneswar, India for his help in tissue preparation and sectioning.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors. No ethical approval is required for this study.


  1. Agronika. (2005). Official publication of government of Odisha. India for fertilizer doses, 541–550.Google Scholar
  2. Bagade, M. A., & Satone, S. R. (2012). An experimental investigation of partial replacement of cement by various percentage of Phosphogypsum in cement concrete. International Journal of Engineering Research and Applications (IJERA) ISSN, 2248–9622.Google Scholar
  3. Bartlett, M. D., Briones, M. J., Neilson, R., Schmidt, O., Spurgeon, D., & Creamer, R. E. (2010). A critical review of current methods in earthworm ecology: from individuals to populations. European Journal of Soil Biology, 46(2), 67–73.CrossRefGoogle Scholar
  4. Bawa, U., Bukar, A., Abdullahi, Y., & Samuel, A. (2016). The role of earthworms in soil structure, nutrients cycle and vermicomposting. ATBU Journal of Science, Technology and Education, 4(2), 149–156.Google Scholar
  5. Bednarska, A. J., Choczyński, M., Laskowski, R., & Walczak, M. (2017). Combined effects of chlorpyriphos, copper and temperature on acetylcholinesterase activity and toxicokinetics of the chemicals in the earthworm Eisenia fetida. Environmental Pollution, 220, 567–576.CrossRefGoogle Scholar
  6. Behera, A. K., Patnaik, A., Mishra, C., & Chhatria, M. (2017). Effect of phosphogypsum on growth and regeneration of earthworm with special reference to Eisenia fetida. PARIPEX – Indian Journal of Research, 6(8), 544–546.Google Scholar
  7. Bianeo, S., Ruggiero, G., Vitti, G. C. and Santus, P. R. (2008) Effects of phosphogypsum and potassium chloride on the nutritional status, production and orangoleptical quality of pineapple fruits. Proc. 3rd Int. Symp. On phosphogypsum. Orlando, Florida, FIPR. Publ. pp. 348–361.Google Scholar
  8. Dittbrenner, N., Schmitt, H., Capowiez, Y., & Triebskorn, R. (2011). Sensitivity of Eisenia fetida in comparison to Aporrectodea caliginosa and Lumbricus terrestris after imidacloprid exposure. Body mass change and histopathology. Journal of Soils and Sediments, 11(6), 1000–1010.CrossRefGoogle Scholar
  9. Ellman, G. L., Courtney, K. D., Andres Jr., V., & Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7(2), 88–95.CrossRefGoogle Scholar
  10. Hach, C. C., Bowden, B. K., Kopelove, A. B., & Brayton, S. V. (1987). More powerful peroxide Kjeldahl digestion method. Association of Official Analytical Chemists Journal (USA).Google Scholar
  11. Ifemeje, J. C., Udedi, S. C., Okechukwu, A. U., Nwaka, A. C., Lukong, C. B., Anene, I. N., … & Ezeude, I. C. (2015). Determination of total protein, superoxide dismutase, catalase activity and lipid peroxidation in soil macro-fauna (earthworm) from Onitsha Municipal Open Waste Dump. Journal of Scientific Research and Reports, 6(5), 394–403.CrossRefGoogle Scholar
  12. Jovanovic, A. M., Durst, S., & Nick, P. (2010). Plant cell division is specifically affected by nitrotyrosine. Journal of Experimental Botany, 61(3), 901–909.CrossRefGoogle Scholar
  13. Kılıç, G. A. (2011). Histopathological and biochemical alterations of the earthworm (Lumbricus terrestris) as biomarker of soil pollution along Porsuk River Basin (Turkey). Chemosphere, 83(8), 1175–1180.CrossRefGoogle Scholar
  14. Lee, B. T., Shin, K. H., Kim, J. Y., & Kim, K. W. (2008). Progress in earthworm ecotoxicology. In Advanced Environmental Monitoring (pp. 248–258). Dordrecht: Springer.CrossRefGoogle Scholar
  15. Lourenço, J. I., Pereira, R. O., Silva, A. C., Morgado, J. M., Carvalho, F. P., Oliveira, J. M., Malta, M. P., Paiva, A. A., Mendo, S. A., & Gonçalves, F. J. (2011). Genotoxic endpoints in the earthworms sub-lethal assay to evaluate natural soils contaminated by metals and radionuclides. Journal of Hazardous Materials, 186(1), 788–795.CrossRefGoogle Scholar
  16. Mays, D. A., & Mortvedt, J. J. (1986). Crop response to soil applications of phosphogypsum 1. Journal of Environmental Quality, 15(1), 78–81.CrossRefGoogle Scholar
  17. Mesić, M., Brezinščak, L., Zgorelec, Ž., Perčin, A., Šestak, I., Bilandžija, D., … & Lisac, H. (2016). The application of phosphogypsum in agriculture. Agriculturae Conspectus Scientificus, 81(1), 7–13.Google Scholar
  18. Mikkelsen, R. U., Fredsted, A., Gissel, H., & Clausen, T. (2004). Excitation-induced Ca2+ influx and muscle damage in the rat: loss of membrane integrity and impaired force recovery. The Journal of Physiology, 559(1), 271–285.CrossRefGoogle Scholar
  19. Muangphra, P. and Gooneratne, R. (2011). Toxicity of commercial neem extract to earthworms (Pheretima peguana). Applied and Environmental Soil Science, 2011 Article ID 925950, 8 pages.Google Scholar
  20. Nayak, S., Mishra, C. S. K., Guru, B. C., & Rath, M. (2011). Effect of phosphogypsum amendment on soil physico-chemical properties, microbial load and enzyme activities. Journal of Environmental Biology, 32, 613–617.Google Scholar
  21. Ohkawa, H., Ohishi, N., & Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95(2), 351–358.CrossRefGoogle Scholar
  22. Olsen, S. R. (1954). Estimation of available phosphorus in soils by extraction with sodium bicarbonate. Washington: United States Department of Agriculture.Google Scholar
  23. Oluah, N. S., Obiezue, R. N. N., Ochulor, A. J., & Onuoha, E. (2010). Toxicity and histopathological effect of atrazine (herbicide) on the earthworm Nsukkadrilus mbae under laboratory conditions. Animal Research International, 7(3), 1287–1293.Google Scholar
  24. Papastefanou, C., Stoulos, S., Ioannidou, A., & Manolopoulou, M. (2006). The application of phosphogypsum in agriculture and the radiological impact. Journal of Environmental Radioactivity, 89(2), 188–198.CrossRefGoogle Scholar
  25. Rao, J. V., & Kavitha, P. (2004). Toxicity of azodrin on the morphology and acetylcholinesterase activity of the earthworm Eisenia foetida. Environmental Research, 96(3), 323–327.CrossRefGoogle Scholar
  26. Rault, M., Mazzia, C., & Capowiez, Y. (2007). Tissue distribution and characterization of cholinesterase activity in six earthworm species. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 147(2), 340–346.CrossRefGoogle Scholar
  27. Samal, S., Sahoo, S., & Mishra, C. S. K. (2017). Morpho-histological and enzymatic alterations in earthworms Drawida willsi and Lampito mauritii exposed to urea, phosphogypsum and paper mill sludge. Chemistry and Ecology, 33(8), 762–776.CrossRefGoogle Scholar
  28. Solé, M., Baena, M., Arnau, S., Carrasson, M., Maynou, F., & Cartes, J. E. (2010). Muscular cholinesterase activities and lipid peroxidation levels as biomarkers in several Mediterranean marine fish species and their relationship with ecological variables. Environment International, 36(2), 202–211.CrossRefGoogle Scholar
  29. Soratto, R. P., & Crusciol, C. A. (2008). Dolomite and phosphogypsum surface application effects on annual crops nutrition and yield. Agronomy Journal, 100(2), 261–270.CrossRefGoogle Scholar
  30. Wacker, W. E., Ulmer, D. D., & Vallee, B. L. (1956). Metalloenzymes and myocardial infarction: malic and lactic dehydrogenase activities and zinc concentrations in serum. New England Journal of Medicine, 255(10), 449–456.CrossRefGoogle Scholar
  31. Walkley, A., & Black, I. A. (1934). An examination of the Degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. Soil Science, 37(1), 29–38.CrossRefGoogle Scholar
  32. Zhang, B. G., Rouland, C., Lattaud, C., & Lavelle, P. (1993). Activity and origin of digestive enzymes in gut of the tropical earthworm Pontoscolex corethrurus [glucidic digestive enzymes; ingested micro-organisms, in vitro tissue culture]. European Journal of Soil Biology (France), 29, 7–11.Google Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Zoology, College of Basic Science and HumanitiesOrissa University of Agriculture and TechnologyBhubaneswarIndia
  2. 2.Post Graduate Department of ZoologyUtkal UniversityBhubaneswarIndia

Personalised recommendations