The LC50 of commercial neem extract (Sadao Thai III containing azadirachtin; NEEM) on filter paper in the earthworm Pheretima peguana at 48？h and 72？h was 3.79 and 3.33？ g？ , respectively. In earthworms exposed to five NEEM concentrations from 0.39 (~10% of 48-h LC50) to 3.13 (~80% of 48-h LC50) g？ , the radial thickness of the epidermis and body wall significantly ( ) decreased, and thickness of intestinal epithelium increased but only at high doses, approximately 25-fold above the concentration permitted for use as an insecticide in field applications (0.09？ g？ ). NEEM significantly ( ) increased the number of binucleated coelomocytes in the micronucleus test (detects chromosomal aberrations) at 3.13？ g？ , approximately 35-fold higher than the recommended dose, but it did not cause coelomocyte DNA single-strand breaks in the comet assay. Thus, NEEM is cytotoxic (increase in binucleates through the inhibition of cytokinesis) but not genotoxic to earthworm coelomocytes. This study demonstrates that the recommended dosage of commercial neem extract as an insecticide in agricultural practices is safe for earthworms. 1. Introduction The most active chemical of neem extract (Sadao Thai III; NEEM) is azadirachtin (Aza), which belongs to the tetranortriterpenoid group of organic compounds. It functions as an ecdysone blocker and a feeding deterrent for some insect pests. There is a perception that NEEM is an environmentally friendly insecticide, so it is often used in high concentrations, and this can lead to a heavy load of NEEM in soil. High NEEM soil concentrations can cause chronic toxicity to nontarget organisms such as crustaceans (Daphnia magna and Hyalella azteca) [1] through leaching from soil into waterways. NEEM has also been shown to induce genotoxicity in rodents [2, 3] and fish [4]. These effects raise concern over the safe use of NEEM as a pesticide in agricultural practices. Although earthworms are often used in terrestrial ecotoxicity evaluation [5], there is little information available on the effects of NEEM on earthworm immune competent cells and histology of the epidermis, skin, body wall, and intestinal lining. In earthworms, coelomocytes are the circulating leukocytes present in the coelomic cavity and play an important role in immune defense. They have been used to study the effect of genotoxicants such as nickel (Ni) and cadmium (Cd) [6, 7] on earthworms. The genotoxicity of NEEM to earthworm coelomocytes has not been reported. DNA damage induced in mammalian and aquatic species by chemical and physical agents can lead to the
References
[1]
I. M. Scott and N. K. Kaushik, “The toxicity of Margosan-O, a product of neem seeds, to selected target and nontarget aquatic invertebrates,” Archives of Environmental Contamination and Toxicology, vol. 35, no. 3, pp. 426–431, 1998.
[2]
K. S. Awasthy, O. P. Chaurasia, and S. P. Sinha, “Genotoxic effects of crude extract of neem (Azadirachta indica) in bone marrow cells of mice,” Cytologia, vol. 60, no. 2, pp. 189–193, 1995.
[3]
P. K. Khan and K. S. Awasthy, “Cytogenetic toxicity of neem,” Food and Chemical Toxicology, vol. 41, no. 10, pp. 1325–1328, 2003.
[4]
P. Chandra and A. R. Khuda-Bukhsh, “Genotoxic effects of cadmium chloride and azadirachtin treated singly and in combination in fish,” Ecotoxicology and Environmental Safety, vol. 58, no. 2, pp. 194–201, 2004.
[5]
M. B. Bouché, “Earthworm species and ecotoxicological studies,” in Ecotoxicology of Earthworms, P. W. Greig-Smith, H. Becker, P. J. Edwards, and F. Heimbach, Eds., pp. 20–35, Intercept, Andover, UK, 1992.
[6]
S. A. Reinecke and A. J. Reinecke, “The comet assay as biomarker of heavy metal genotoxicity in earthworms,” Archives of Environmental Contamination and Toxicology, vol. 46, no. 2, pp. 208–215, 2004.
[7]
F. Fourie, S. A. Reinecke, and A. J. Reinecke, “The determination of earthworm species sensitivity differences to cadmium genotoxicity using the comet assay,” Ecotoxicology and Environmental Safety, vol. 67, no. 3, pp. 361–368, 2007.
[8]
R. Schlegel and J. T. MacGregor, “The persistence of micronuclei in peripheral blood erythrocytes: detection of chronic chromosome breakage in mice,” Mutation Research, vol. 104, no. 6, pp. 367–369, 1982.
[9]
I. Udroiu, “The micronucleus test in piscine erythrocytes,” Aquatic Toxicology, vol. 79, no. 2, pp. 201–204, 2006.
[10]
M. A. Farah, B. Ateeq, and W. Ahmad, “Antimutagenic effect of neem leaves extract in freshwater fish, Channa punctatus evaluated by cytogenetic tests,” Science of the Total Environment, vol. 364, no. 1–3, pp. 200–214, 2006.
[11]
A. V. Pavlov, M. A. Gansburgskii, A. N. Gansburgskii, M. V. Shashkina, and T. L. Miro, “The use of micronucleus test for detection of genotoxic damage to the thyroid gland,” Bulletin of Experimental Biology and Medicine, vol. 141, no. 1, pp. 99–102, 2006.
[12]
M. Toyoshima, K. Ohno, T. Katsumoto, H. Maki, and K. Takeshita, “Cellular senescence of angiofibroma stroma cells from patients with tuberous sclerosis,” Brain and Development, vol. 21, no. 3, pp. 184–191, 1999.
[13]
A. Salehzadeh, A. Akhkha, W. Cushley, R. L. P. Adams, J. R. Kusel, and R. H. C. Strang, “The antimitotic effect of the neem terpenoid azadirachtin on cultured insect cells,” Insect Biochemistry and Molecular Biology, vol. 33, no. 7, pp. 681–689, 2003.
[14]
F. L. Martin, S. Venitt, P. L. Carmichael et al., “DNA damage in breast epithelial cells: detection by the single-cell gel (comet) assay and induction by human mammary lipid extracts,” Carcinogenesis, vol. 18, no. 12, pp. 2299–2305, 1997.
[15]
R. R. Tice, E. Agurell, D. Anderson et al., “Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing,” Environmental and Molecular Mutagenesis, vol. 35, no. 3, pp. 206–221, 2000.
[16]
F. L. Martin, T. G. Piearce, A. Hewer, D. H. Phillips, and K. T. Semple, “A biomarker model of sublethal genotoxicity (DNA single-strand breaks and adducts) using the sentinel organism Aporrectodea longa in spiked soil,” Environmental Pollution, vol. 138, no. 2, pp. 307–315, 2005.
[17]
C. A. Edward and J. R. Lofty, Biology of Earthworms, Chapman and Hall, London, UK, 1st edition, 1972.
[18]
A. Amaral, M. Soto, R. Cunha, I. Marigómez, and A. Rodrigues, “Bioavailability and cellular effects of metals on Lumbricus terrestris inhabiting volcanic soils,” Environmental Pollution, vol. 142, no. 1, pp. 103–108, 2006.
[19]
E. fischer and L. Molnár, “Environmental aspects of the chloragogenous tissue of earthworms,” Soil Biology and Biochemistry, vol. 24, no. 12, pp. 1723–1727, 1992.
[20]
A. J. Morgan, M. P. Turner, and J. E. Morgan, “Morphological plasticity in metal-sequestering earthworm chloragocytes: morphometric electron microscopy provides a biomarker of exposure in field populations,” Environmental Toxicology and Chemistry, vol. 21, no. 3, pp. 610–618, 2002.
[21]
N. Kaushik, “Determination of azadirachtin and fatty acid methyl esters of Azadirachta indica seeds by HPLC and GLC,” Analytical and Bioanalytical Chemistry, vol. 374, no. 7-8, pp. 1124–1199, 2002.
[22]
W. Kwankua, S. Sengsai, C. Kuleung, and N. Euawong, “Sunlight decreased genotoxicity of azadirachtin on root tip cells of Allium cepa and Eucrosia bicolor,” Ecotoxicology and Environmental Safety, vol. 73, no. 5, pp. 949–954, 2010.
[23]
G. S. Eyambe, A. J. Goven, L. C. Fitzpatrick, B. J. Venables, and E. L. Cooper, “A non-invasive technique for sequential collection of earthworm (Lumbricus terrestris) leukocytes during subchronic immunotoxicity studies,” Laboratory Animals, vol. 25, no. 1, pp. 61–67, 1991.
[24]
P. Brousseau, N. Fugère, J. Bernier et al., “Evaluation of earthworm exposure to contaminated soil by cytometric assay of coelomocytes phagocytosis in Lumbricus terrestris,” Soil Biology and Biochemistry, vol. 29, no. 3-4, pp. 681–684, 1997.
[25]
N. P. Singh, M. T. McCoy, R. R. Tice, and E. L. Schneider, “A simple technique for quantitation of low levels of DNA damage in individual cells,” Experimental Cell Research, vol. 175, no. 1, pp. 184–191, 1988.
[26]
J. Homa, E. Olchawa, S. R. Stürzenbaum, A. J. Morgan, and B. Plytycz, “Early-phase immunodetection of metallothionein and heat shock proteins in extruded earthworm coelomocytes after dermal exposure to metal ions,” Environmental Pollution, vol. 135, no. 2, pp. 275–280, 2005.
[27]
A. Anuradha, R. S. Annadurai, and L. S. Shashidhara, “Actin cytoskeleton as a putative target of the neem limonoid azadirachtin A,” Insect Biochemistry and Molecular Biology, vol. 37, no. 6, pp. 627–634, 2007.
[28]
Z. Y. Wang, L. Z. Zou, B. L. Fan, and Y. K. Peng, “Abnormal metaphase cell division induced by microtubules depolymerization and photosystem II inhibiting herbicides,” Cytologia, vol. 71, no. 3, pp. 289–295, 2006.
[29]
J. V. Rao, P. Kavitha, and A. P. Rao, “Comparative toxicity of tetra ethyl lead and lead oxide to earthworms, Eisenia fetida (Savigny),” Environmental Research, vol. 92, no. 3, pp. 271–276, 2003.
[30]
N. C. Reddy and J. V. Rao, “Biological response of earthworm, Eisenia foetida (Savigny) to an organophosphorous pesticide, profenofos,” Ecotoxicology and Environmental Safety, vol. 71, no. 2, pp. 574–582, 2008.
