Oxidative Damage and Genetic Toxicity Induced by DBP in Earthworms (Eisenia fetida)

  • Guanying Wang
  • Jun Wang
  • Lusheng Zhu
  • Jinhua Wang
  • Hengzhou Li
  • Yizhang Zhang
  • Wenjun Liu
  • Jianpeng Gao


Di-n-butyl phthalate (DBP) is one of the most ubiquitous plasticizers used worldwide. However, it has negatives effects on the soil, water, atmosphere, and other environmental media and can cause serious pollution. According to the artificial soil test and previous studies, this study was conducted to evaluate the toxicity of earthworms induced by DBP at different concentrations (0, 0.1, 1.0, 10, and 50 mg kg−1) on the 7th, 14th, 21st, and 28th days of exposure. The variations in the antioxidant activities of enzymes, such as catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glutathione-S-transferase (GST), in the amounts of malondialdehyde (MDA) and reactive oxygen species (ROS) and in the amount of DNA damage were measured to evaluate the toxic impact of DBP in earthworms. Upon exposure to DBP, the SOD, CAT, POD, and GST activities were significantly increased, with the exception of the 0.1 mg kg−1 treatment dose. High concentrations of DBP (10 and 50 mg kg−1) induced superfluous ROS to be produced and caused the MDA content to increase significantly. Therefore, we proposed that DBP led to DNA damage in earthworm coelomocytes in a dose-dependent manner, which means that DBP is a source of oxidative damage and genetic toxicity in earthworms.



This study under the auspices of the National Key Research and Development Project of China (Grant number 2016YFD0800304) and Natural Science Foundation of Shandong (Grant number ZR2017MD023).

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals (Eisenia fetida) were followed.


  1. Alam MS, Ohsako S, Matsuwaki T, Zhu XB, Tsunekawa N, Kanai Y, Sone H, Tohyama C, Kurohmaru M (2010) Induction of spermatogenic cell apoptosis in prepubertal rat testes irrespective of testicular steroidogenesis: a possible estrogenic effect of di(n-butyl) phthalate. Reproduction 139(2):427–437CrossRefGoogle Scholar
  2. Beauchamp C, Fridovicich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–278CrossRefGoogle Scholar
  3. Clewell RA, Kremer JJ, Williams CC, Campbell JL, Sochaski MA, Andersen ME, Borghoff SJ (2009) Kinetics of selected di-n-butyl phthalate metabolites and fetal testosterone following repeated and single administration in pregnant rats. Toxicology 255(1–2):80–90CrossRefGoogle Scholar
  4. Coleman DC, Ingham ER (1988) Carbon, nitrogen, phosphorus and sulfur cycling in terrestrial ecosystems. Biogeochemistry 5:3–6CrossRefGoogle Scholar
  5. Cooke MS, Evans MD, Dizdaroglu M, Lunec J (2003) Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J 17(10):1195–1214CrossRefGoogle Scholar
  6. Daiem MMA, Rivera-Utrilla J, Ocampo-Perez R, Mendez-Diaz JD, Sanchez-Polo M (2012) Environmental impact of phthalic acid esters and their removal from water and sediments by different technologies–a review. J Environ Manag 109:164–178CrossRefGoogle Scholar
  7. Du L, Li G, Liu M, Li Y, Yin S, Zhao J (2015) Biomarker responses in earthworms (eisenia fetida) to soils contaminated with di-n-butyl phthalates. Environ Sci Pollut Res 22(6):4660–4669CrossRefGoogle Scholar
  8. Gao J, Chi J (2015) Biodegradation of phthalate acid esters by different marine microalgal species. Mar Pollut Bull 99(1–2):70–75CrossRefGoogle Scholar
  9. Ge W, Yan S, Wang J, Zhu L, Chen A, Wang J (2015) Oxidative stress and DNA damage induced by imidacloprid in zebrafish (danio rerio). J Agric Food Chem 63(6):1856–1862CrossRefGoogle Scholar
  10. Greenwald RA (1987) Handbook of methods for oxygen radical research. Free Radical Biol Med 3(2):161Google Scholar
  11. Guo X, Wang L, Wang X, Liu H (2013) Occurrence and environmental risk assessment of PAEs in Weihe river near Xi’an city, China. Water Sci Technol 67(5):948–958CrossRefGoogle Scholar
  12. Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione s-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139Google Scholar
  13. Han Y, Zhu L, Wang J, Wang J, Xie H, Zhang S (2014) Integrated assessment of oxidative stress and DNA damage in earthworms (Eisenia fetida) exposed to azoxystrobin. Ecotoxicol Environ Saf 107:214–219CrossRefGoogle Scholar
  14. Hassanzadeh N, Esmaili SA, Khodabandeh S, Bahramifar N (2014) Occurrence and distribution of two phthalate esters in the sediments of the Anzali wetlands on the coast of the Caspian Sea (Iran). Mar Pollut Bull 89(1–2):128–135CrossRefGoogle Scholar
  15. Jayawardena I, Godakumbura PI, Prashantha MA (2016) Migration of BTEX and phthalates from natural rubber latex balloons obtained from the Sri Lankan market. Springerplus 5:20CrossRefGoogle Scholar
  16. Jiang JT, Zhong C, Zhu YP, Xu DL, Wood K, Sun WL, Li EH, Liu ZH, Zhao W, Ruan Y, Xia SJ (2016) Prenatal exposure to di-n-butyl phthalate (DBP) differentially alters androgen cascade in undeformed versus hypospadiac male rat offspring. Reprod Toxicol 61:75–81CrossRefGoogle Scholar
  17. Kochba J, Lavee S, Spiegel-Roy P (1977) Differences in peroxidase activity and soenzymes in embryogenic and non-embryogenic ‘Shamouti’ orange ovular callus lines. Plant Cell Physiol 18:463–467Google Scholar
  18. Kosmehl T, Hallare AV, Braunbeck T, Hollert H (2008) DNA damage induced by genotoxicants in zebrafish (Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from laguna lake (the Philippines) as measured by the comet assay. Mutat Res 650(1):1–14CrossRefGoogle Scholar
  19. Lawler J (2003) Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle. Free Radic Biol Med 35(1):9–16CrossRefGoogle Scholar
  20. Liao CS, Yen JH, Wang YS (2009) Growth inhibition in chinese cabbage (Brassica rapa var. Chinensis) growth exposed to di-n-butyl phthalate. J Hazard Mater 163(2–3):625–631CrossRefGoogle Scholar
  21. Liu W, Zhu LS, Wang J, Wang JH, Xie H, Song Y (2009) Assessment of the genotoxicity of endosulfan in earthworm and white clover plants using the comet assay. Arch Environ Contam Toxicol 56(4):742–746CrossRefGoogle Scholar
  22. Liu SB, Ma Z, Sun WL, Sun XW, Hong Y, Ma L, Qin C, Stratton HJ, Liu Q, Jiang JT (2012) The role of androgen-induced growth factor (FGF8) on genital tubercle development in a hypospadiac male rat model of prenatal exposure to di-n-butyl phthalate. Toxicology 293(1–3):53–58CrossRefGoogle Scholar
  23. Lu Y, Tang F, Wang Y, Zhao J, Zeng X, Luo Q, Wang L (2009) Biodegradation of dimethyl phthalate, diethyl phthalate and di-n-butyl phthalate by rhodococcus sp. L4 isolated from activated sludge. J Hazard Mater 168(2–3):938–943CrossRefGoogle Scholar
  24. Massicotte JP (1994) Oxidative processes as indicators of chemical stress in marine bivalves. Aquat Ecosyst Health 3:101–111CrossRefGoogle Scholar
  25. Matsumoto M, Hirata-Koizumi M, Ema M (2008) Potential adverse effects of phthalic acid esters on human health: a review of recent studies on reproduction. Regul Toxicol Pharmacol 50(1):37–49CrossRefGoogle Scholar
  26. Mishra PC, Dash MC (1980) Digestive enzymes of some earthworms. Experientia 36(10):1156–1157CrossRefGoogle Scholar
  27. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7(9):405–410CrossRefGoogle Scholar
  28. Motohashi M, Wempe MF, Mutou T, Takahashi H, Kansaku N, Ikegami M, Inomata T, Asari M, Wakui S (2016) Male rats exposed in utero to di(n-butyl) phthalate: age-related changes in leydig cell smooth endoplasmic reticulum and testicular testosterone-biosynthesis enzymes/proteins. Reprod Toxicol 59:139–146CrossRefGoogle Scholar
  29. OECD (1984) Test 207: Earthworm, acute toxicity tests. In: Organization for economic cooperation and development. OECD guidelines for testing of chemicals. Organization for Economic Cooperation and Development (OECD). Paris, FranceGoogle Scholar
  30. OECD (2004) Test 202: Daphnia sp-acute immobilisation test. In: Organization for testing of chemicals. Organization for the Economic Cooperation and Development, Paris, FranceGoogle Scholar
  31. Pavlica M, Klobucar GIV, Mojaš N, Erben R, Papeš D (2009) Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutat Res 490:209–214CrossRefGoogle Scholar
  32. Pérez-Feás C, Barciela-Alonso MC, Bermejo-Barrera P (2011) Presence of phthalates in contact lens and cleaning solutions. Microchem J 99(1):108–113CrossRefGoogle Scholar
  33. Reineme P, Prade L, Hof P, Neuefeind T, Huber R, Zettl R, Palme K, Schell J, Koelln I, Bartunik HD, Bieseler B (1996) Three-dimensional structure of glutathione s-transferase from arabidopsis thaliana at 2.2 a resolution: structural characterization of herbicide-conjugating plant glutathione s-transferases and a novel active site architecture. J Mol Biol 255:289–309CrossRefGoogle Scholar
  34. Seitz N, Bottcher M, Keiter S, Kosmehl T, Manz W, Hollert H, Braunbeck T (2008) A novel statistical approach for the evaluation of comet assay data. Mutat Res 652(1):38–45CrossRefGoogle Scholar
  35. Sen N, Liu X, Craig ZR (2015) Short term exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in young CD-1 mice. Reprod Toxicol 53:15–22CrossRefGoogle Scholar
  36. Sforzini S, Boeri M, Dagnino A, Oliveri L, Bolognesi C, Viarengo A (2012) Genotoxicity assessment in Eisenia andrei coelomocytes: a study of the induction of DNA damage and micronuclei in earthworms exposed to B[a]P- and TCDD-spiked soils. Mutat Res 746(1):35–41CrossRefGoogle Scholar
  37. Sha Y, Xia X, Yang Z, Huang GH (2007) Distribution of PAEs in the middle and lower reaches of the yellow river, China. Environ Monit Assess 124(1–3):277–287CrossRefGoogle Scholar
  38. Shugart LR (2000) DNA damage as a biomarker of exposure. Ecotoxicology 9:329–340CrossRefGoogle Scholar
  39. Singh NP, Mccoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191CrossRefGoogle Scholar
  40. Singh S, Eapen S, D’souza SF (2006) Cadmium accumulation and its influence on lipid peroxidation and antioxidative system in an aquatic plant, Bacopa monnieri L. Chemosphere 62(2):233–246CrossRefGoogle Scholar
  41. Song Y, Zhu LS, Wang J, Wang JH, Liu W, Xie H (2009) DNA damage and effects on antioxidative enzymes in earthworm (Eisenia fetida) induced by atrazine. Soil Biol Biochem 41(5):905–909CrossRefGoogle Scholar
  42. Sun LW, Qu MM, Li YQ, Wu YL, Chen YG, Kong ZM, Liu ZT (2004) Toxic effects of aminophenols on aquatic life using the zebrafish embryo test and the comet assay. Bull Environ Contam Toxicol 73(4):628–634CrossRefGoogle Scholar
  43. Swan SH (2008) Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans. Environ Res 108(2):177–184CrossRefGoogle Scholar
  44. Teil MJ, Blanchard M, Chevreuil M (2006) Atmospheric fate of phthalate esters in an urban area (Paris-France). Sci Total Environ 354(2–3):212–223CrossRefGoogle Scholar
  45. US EPA (1991) Risk Assessment Guidance for Superfund (RAGS): Volume I-Humman Health Evaluation Manual(HHEM) (Part B, Development of Risk-Based Preliminary Remediation Goals). Office of Emergency and Remedial Response, Washington DC, EPA/540/R-92/003, OSWER Directive 9285.7-01B, NTIS PB92-963333.r)Google Scholar
  46. Valavanidis A, Vlahogianni T, Dassenakis M, Scoullos M (2006) Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol Environ Safe 64(2):178–189CrossRefGoogle Scholar
  47. Vitali M, Guidotti M, Macilenti G, Cremisini C (1997) Phthalate esters in freshwaters as markers of contamination sources—a site study in Italy. Environ Int 23(3):337–347CrossRefGoogle Scholar
  48. Wang XH (2010) Effects of DBP/DEHP on soil microbial biomass carbon and enzyme in single and complex pollution with Pb. dissertation. University of Dongbei NormalGoogle Scholar
  49. Wang XF, Xing ML, Shen Y, Zhu X, Xu LH (2006) Oral administration of Cr(VI) induced oxidative stress, DNA damage and apoptotic cell death in mice. Toxicology 228(1):16–23CrossRefGoogle Scholar
  50. Wang JH, Zhu LS, Meng Y, Wang J, Xie H, Zhang QM (2012) The combined stress effects of atrazine and cadmium on the earthworm Eisenia fetida. Environ Toxicol Chem 31(9):2035–2040CrossRefGoogle Scholar
  51. Winston GW, Giulio RTD (1991) Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat Toxicol 19(2):137–161CrossRefGoogle Scholar
  52. Xie Z, Wang J, Dai F, Jin X, Wu K, Chen Q, Wang Y (2016) Effects of maternal exposure to di-n-butyl phthalate during pregnancy and breastfeeding on ovarian development and function of F1 female rats. Environ Toxicol Pharmacol 43:38–43CrossRefGoogle Scholar
  53. Xue Y, Gu X, Wang X, Sun C, Xu X, Sun J, Zhang B (2009) The hydroxyl radical generation and oxidative stress for the earthworm Eisenia fetida exposed to tetrabromobisphenol A. Ecotoxicology 18(6):693–699CrossRefGoogle Scholar
  54. Yan S, Wang J, Zhu L, Chen A, Wang J (2015) Toxic effects of nitenpyram on antioxidant enzyme system and DNA in zebrafish (Danio rerio) livers. Ecotoxicol Environ Safe 122:54–60CrossRefGoogle Scholar
  55. Yang CF, Wang CC, Chen CH (2013) Di-n-butyl phthalate removal using mixed cultures in batch reactors. Int Biodeterior Biodegrad 85:587–591CrossRefGoogle Scholar
  56. Zhang QF, Li YY, Pang CH, Lu CM, Wang BS (2005) Nacl enhances thylakoid-bound sod activity in the leaves of c3 halophyte Suaeda salsa L. Plant Sci 168(2):423–430CrossRefGoogle Scholar
  57. Zhang Q, Zhu L, Wang J, Xie H, Wang J, Han Y, Yang J (2013) Oxidative stress and lipid peroxidation in the earthworm Eisenia fetida induced by low doses of fomesafen. Environ Sci Pollut Res 20(1):201–208CrossRefGoogle Scholar
  58. Zhang Y, Du N, Wang L, Zhang H, Zhao J, Sun G, Wang P (2015) Physical and chemical indices of cucumber seedling leaves under dibutyl phthalate stress. Environ Sci Pollut Res 22(5):3477–3488CrossRefGoogle Scholar
  59. Zheng LP, Feng YH, Zhao X, Xu J, Lin YS (2010) Toxicity effects of chlordane and mirex contaminated soil on earthworm. J Agro-Environ Sci 29(10):1924–1929Google Scholar
  60. Zhou Q, Mrowietz U, Rostami-Yazdi M (2009) Oxidative stress in the pathogenesis of psoriasis. Free Radic Biol Med 47(7):891–905CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Guanying Wang
    • 1
    • 2
    • 3
  • Jun Wang
    • 1
  • Lusheng Zhu
    • 1
  • Jinhua Wang
    • 1
  • Hengzhou Li
    • 1
  • Yizhang Zhang
    • 3
  • Wenjun Liu
    • 1
  • Jianpeng Gao
    • 1
  1. 1.College of Resources and Environment, Key Laboratory of Agricultural EnvironmentShandong Agricultural UniversityTai′anChina
  2. 2.College of Water SciencesBeijing Normal UniversityBeijingChina
  3. 3.Chinese Research Academy of Environmental SciencesBeijingChina

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