International Journal of Environmental Protection

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Genotoxic Endpoints in Allium cepa and Clarias gariepinus Exposed to Textile Effluent
Victoria A.Oriaku1, Olubunmi A.Otubanjo2, Adeolu O. Aderemi3, Adebayo A. Otitoloju4
1,3,4 2

Ecotoxicology unit, Department of Zoology. University of Lagos, Nigeria Parasitology unit, Department of Zoology. University of Lagos, Nigeria adannaoriaku@gmail.com1,ootubanjo@unilag.edu.ng2 bodeoludunks@yahoo.com3,aotitoloju@unilag.edu.ng4

Abstract- The genotoxic effects of textile effluent were investigated in Clarias gariepinus and Allium cepa using the micronucleus assay and induction of chromosomal aberration in respectively. The aim of the study is to provide genotoxic endpoints that can serve as biomarkers of exposure to textile effluents in the environment. The physico-chemical characterization of the effluent revealed high levels of chemical oxygen demand (COD), biological oxygen demand (BOD) and total suspended solids (TSS) above the standards set by regulatory body. Exposure of Clarias gariepinus to sub-lethal concentrations of effluent resulted in a dose-dependent and significant (P<0.05) increase in the formation of micronuclei and nuclear anomalies in blood erythrocytes. The textile effluent also caused a significant (P<0.5) reduction in mean root length and increased chromosomal aberrations such as vagrant, sticky chromosomes, binucleus and c-tumors in exposed Allium cepa compared to control. The results obtained from this study indicate the textile effluent being discharged into the Lagos lagoon have genotoxic potential and is capable of causing significant ecological disruption in the receiving environment. Keywords--textile effluent, gariepinus, Allium cepa genotoxic effects, Clarias

a wide range of compounds [10]. The Allium cepa test is a simple, sensitive and rapid bioassay that has been widely used as a standard for biomonitoring of environmental contaminants using various genotoxicity parameters [9]. Based on the significance of the micronucleus test and Allium test this study is aimed at assessing the genotoxic effects of textile effluents on a plant, A.cepa and an animal, C.gariepinus. II.
MATERIALS AND METHODS

A. Collection of Effluent Effluent was collected at different outlets discharge from a creek receiving direct effluent discharge from a textile mill at Ikorodu, Lagos, Nigeria. The effluent kept in plastic containers, were mixed and stored at 4C prior to analysis. B. Physical and Chemical Characterization The physico-chemical properties of the effluent sample were determined in accordance with standard analytical methods. C. Test Organisms Fish: Clarias gariepinus (diploid chromosome 2n = 56) was chosen for this study because of its common availability in most fish farms in Nigeria. Clarias gariepinus juveniles of similar sizes (12-14cm) were purchased from a commercial fish farm. The juveniles were kept in glass tanks in the laboratory and allowed to acclimatize for at least 7 days to laboratory conditions. Onion Bulb: Purple strain of Allium cepa (2n = 16) of size 2.0 to 2.5cm and weigh 2 to 4g was used for the Allium cepa test. The onion was purchased from the local market. The onion roots were intact and dry, free from decay and other forms of mechanical damage. D. Micronuclei Assay and Nuclear Abnormalities in C. gariepinus. Preliminary screening was carried out to determine the appropriate concentration range for effluent, prior to experiment. At the end of 96h, the concentration in which all fishes stay alive or no mortality was observed- LC50) was determined at 1.6ml L-1. The concentrations 0.16 and 0.016 ml L-1(1/10th and 1/100th respectively of the LC50) were selected for the experiment. Fishes were placed in tank containing dechlorinated tap water (control) and two different concentrations of textile effluents (0.16 and 0.016 ml L-1). For the micronucleus test, blood samples were obtained from

I. INTRODUCTION The textile industry is one of the prominent industries in Nigeria, with attendant indiscriminate discharge of effluents into the surrounding water bodies. The textile industry poses severe environmental problems mainly due to the large volumes of wastewater it generates, and the complex composition of the effluents produced [5]. The chemical reagents used are very diverse in chemical composition, ranging from inorganic compounds to polymers and organic products such as dyes, plasticizers, detergents [22]. These effluents contain selected important environmental pollutants that tend to accumulate in organisms, and persist in the environment due to their chemical stability or poor biodegradability. In developing countries, due to the high cost of treatment, these effluents are often not treated prior to discharge into the environment. The introduction of such effluents into the aquatic system has caused several disruptions to aquatic productivity and eventually leads to some physiological dysfunction in the resident organisms [14]. A variety of biomarkers and bio-assays can be used in the laboratory and field studies to determine the effects of genotoxic pollutants on biotic systems, these include the presence of DNA adducts, chromosomal aberrations and DNA strand breaks. The micronucleus test is widely applied, due to its simplicity, reliability, sensitivity and proven suitability for fish species [11]. It detects both clastogenic and aneugenic effects and therefore can detect the genotoxicity of DOI 10.5963/IJEP0105007

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the caudal sinus on the 5th, 10th, and 15th day of exposure. Twenty-five (25) fishes were used for the assay which was done in duplicates (5 fishes per replicate for each concentration and 5 for the control). Peripheral blood samples were obtained from the caudal sinus using a syringe. Blood was smeared immediately on clean grease free microscope slides, air dried for 12 hrs and then fixed in absolute methanol for 20min. Each slide was stained with 5% Giemsa solution for 30mins. Three slides were prepared for each fish from the experimental and control experiments were viewed microscopically. Small, nonrefractile, circular or ovoid chromatin bodies showing the same staining pattern as the main nucleus were considered as micronuclei (MN) [13]. Studies from [4] classify nuclear abnormalties into blebbed nuclei (BL), lobed nuclei (LB), notched nuclei (NT) and binuclei (BN). The frequencies of micronuclei and other nuclear lesions were expressed per 1000 cells (0/00). E. Root Growth Inhibition and Chromosomal Aberration in A. cepa The assay was carried out using a plastic tube (diameter, 2.3 cm; length, 7.8 cm) in a rack. Dechlorinated water was used as control and for dilution of industrial effluents. The yellow shallows and dry bottom plate inside the root primordial of A. cepa were carefully removed prior to the test. The toxicity assay is performed as a 96 h semi static exposure test, and six concentrations (10, 20, 30, 40, 50 and 100%) of the textile effluents are used. Every 24 h the test solutions are replaced with fresh solutions and the length of the root bundle is measured. At the termination of the exposure, one onion (out of six) with the poorest growth is discarded and the length of the root bundle is measured for the remaining five onions. Growth inhibition was estimated as EC50 (the effective concentration of a chemical producing 50% of the total effect). For each of the concentration there were 4 replicates. The root tip of each replicate was selected and fixed in 4 different fixative bottles using acetic acid. From each bottle containing fixed roots, 5 microscopic slides were made using the aceto orcein squash method. From each concentration, 8 microscopic slides were prepared. A total of 48 slides were made. The slides were viewed microscopically for mitotic or cytological aberrations and micrographs were taken. The mitotic index (MI) was determined by the examination of 500 cells per concentration (100 cells per slide). Characterization of mitosis and chromosomal aberrations were scored in 100 cells per slide. F. Statistical Analysis Fish (Clarias gariepinus): Probit analysis was used to determine the 96 h LC50 value of the effluent. All data were expressed as meansSE. A twoway analysis of variance (ANOVA) was used to determine the significance of micronucleus tests. Bonferroni post tests were used to compare frequency of nuclear abnormalities and micronucleus between control and treatment groups. Statistical tests were performed using the GraphPad Prism for PC computers. 1) Onion (Allium cepa):

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The EC50 and regression equation were determined from a plot of root length as a percentage of control against the sample concentrations, by using a Microsoft Excel computer program. Pearson correlation analysis was carried out to test for significant relationship (positive or negative) between the root length and effluent concentrations. Analysis of Variance (ANOVA) and Student Newman Keuls (SNK) tests were used to test for significant differences in the mean root lengths of A. cepa exposed to different concentrations of textile effluents. The tests were carried out at 5% significant level. The analysis was performed using SPSS 10.0 computer program [20]. III. RESULTS A. Physico-Chemical Characterisation of Textile Effluent The results of the physico-chemical characterization of textile effluent revealed higher values of most parameters than the standards set by environmental protection agency (Table 1).
TABLE I PHYSICO-CHEMICAL CHARACTERISTICS OF THE TEXTILE EFFLUENT Parameters Concentration Effluent (mg/L) BOD COD TSS Oil and grease pH Cr Cu Phenol NH4+ Free chlorine 152 319 1160 612 6.9 1.20 0.02 Not detected 0.50 Not detected Standards(mg/L) 20 8 30 10 6-9 <1 <1 0.01 1.0

TABLE II MEANSE OF MICRONUCLEUS FREQUENCIES (0/00) IN ERYTHROCYTES OF C. GARIEPINUS EXPOSED TO DIFFERENT CONCENTRATIONS OF TEXTILE EFFLUENT

Concentration ml L
-1

Time of exposure

Day 5 Control 0.16 0.016 0.500.28 4.750.62 2.500.28

Day 10 0.250.25 2.750.47 1.250.47

Day 15 0.250.25 1.500.28 0.750.47

B. Effect of Textile Effluents on Micronucleus Formation and Nuclear Abnormalities. An LC50 value of 1.639ml L-1 was obtained at the end of the 96h acute toxicity assay. It was observed that the micronucleus frequencies increased dose-dependent as they decreased time-dependently (Table 2). Formation of micronucleus were found to be significant (P<0.01) and (P<0.001) at day 5 and day 10 respectively.

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Similarly, frequencies of nuclear abnormality in erythrocytes were also positively dose-dependent and negatively time-dependent (Fig.1), 0.16ml L-1 of effluent was significant (P<0.01) and (P<0.05) at days 5 and 10 respectively. Anomalies of the erythrocytes are presented in Fig. 2.
6
Concentration (ml/L)

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1/10 Textile Effluent 1/100 Textile Effluent Control

0
10 Da y Da y Da y 15 5

Fig. 1 Changes in the frequencies of nuclear abnormalities in erythrocytes

Fig. 4 Various chromosomal aberrations. a) abnormal metaphase (attached chromosome) (50% textile effluent); b) binucleus (100% textile effluent); c) vagrant chromosome (10% textile effluent); d) Sticky chromosomes (100% textile effluent)

TABLE III MEAN ROOT LENGTH OF A. CEPA EXPOSED TO DIFFERENT CONCENTRATIONS OF TEXTILE EFFLUEN Textile effluent concentration (%) Mean root length (cm)

a
Control (0) 10 20 4.60E 2.70D 1.98C 1.65C 1.30B 0.83A 0.33A

b
Fig. 2 Various nuclear abnormalities. a) micronucleus; b) notched nucleus
110 100 90 80 70
Root Length (%)

30 40 50 100

Means with the same superscript letter (A, B, C, D and E) along the column are not significantly different in the SNK test at P<0.05

60 50 40 30 20 10 0 0 10 20 30 40 50 60 70 80 90 100 110 y = 68.92 - 0.76x r = 0.83

EC50= 25
EC 2

Concentration (%)

Fig. 3 Growth inhibition of Allium cepa roots exposed to textile effluent

C. Effect of Textile Effluent on Root Growth and Chromosomal Aberration in A. cepa The estimated EC50 of A. cepa exposed to textile effluent was 25% (Fig. 3). Reduction in root lengths was observed in onion root tips exposed to all concentrations of textile effluent. The root growth inhibition was concentration dependent as shown in Fig. 3. Statistical analysis revealed that the root length of A. cepa exposed to control was significant (P<0.05) from the root length of A. cepa exposed to all other concentrations (Table 3). Results of microscopic effects and chromosomal aberrations of A. cepa roots exposed to textile effluent are summarized in Table 4 and Fig. 4. There was rapid decrease in the mitotic index (MI) with increasing concentration of the effluent. Five hundred mitoses could not be attained for chromosome screening compared to control due to the effects of effluent on cells. Analysis of the chromosomes showed that the effluent induced chromosomal

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TABLE IV EFFECTS OF TREATMENT WITH DIFFERENT CONCENTRATIONS OF TEXTILE EFFLUENT Number Number of stickin cVagrant Bridges binuclei Multipolar of cell dividing cell ess mitosis fragment Anaphase 500 447 426 388 315 259 237 42[P16M10A9 T7] 39[P12M7A11 T9] 27[P7M11A5T 4] 21[P5M10A2T 4] 17 [P5M4A3T5] 13 [P2M4A4T3] 12 [P0M3A7T2] 0 7 4 3 4 3 2 0 0 0 2 1 0 0 0 5 5 2 2 4 6 0 3 8 7 4 4 3 0 0 0 2 0 0 4 0 0 0 0 0 0 1

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Concentr ation (%) Control (0) 10 20 30 40 50 100

Mitotic index 8.4 8.7 6.3 5.4 5.3 5.0 5.0

Attached

TA(%) SE 3.80.74 4.50.58 4.60.60 3.80.50 4.20.48 6.80.8

0 2 2 1 1 0 0

Mitotic index (MI) was calculated as number of dividing cells/number of cells per 500 cells (100 from each slide).P, M, A and T represent Prophase, Metaphase, Anaphase and Telophase respectively. TA and SE represent Total aberration and Standard error respectively.

aberrations significantly when compared to the control. In the control no aberration was recorded in the chromosome of A. cepa. IV. DISCUSSION The physicochemical properties of the effluent obtained were mostly higher than the Federal Environmental Protection Agency (FEPA) safe limit for effluents discharged into water bodies and this resounds the belief that industry operators within the country disregard safe limits set by the regulatory agency. All these point to a lag in the mechanism of effluent quality assessment, mitigation and enforcement on the part of the regulators. Oil and grease concentration was over 60 times the set limit while total suspended solid was also about 39 times above the limit. These raise concerns about the health of the Lagos Lagoon ecosystem where these effluents eventually deposits. Further concern is raised by the tendency of some metals such as chromium and copper contained in the effluent to bioaccumulate in the river beds and hence enter into aquatic and sometimes terrestrial food chains where they may result in deleterious effects. The high level of biological oxygen demand (BOD) (152mg/l) reported can be linked to the nature of the raw materials used and the waste generated by various processes in the industry. Textile industry wastes are known to contain organic materials/compounds such as dyes, nutrients, molasses, fibres, cellulose and esters [12]. However the high level of BOD reported is not unusual; some earlier authors have reported high levels in the principal media of the Lagos. Lagoon as well as effluents destined for discharge in the Lagoon [2]. These have been found to have far reaching effects in the benthic community and food chain structure of the Lagoon and associated water bodies [3]. The high level of chemical oxygen demand (4x higher than the FEPA safe limit) recorded in the effluent indicates that there is high chemical input in the effluent. High BOD values results in the depletion of available oxygen for aquatic residents, while high amount of COD in effluent is toxic to biological life. Heavy metals like cadmium and mercury were not detected and this is in consonance with earlier findings in the Lagos Lagoon and water bodies elsewhere in the country where the DOI 10.5963/IJEP0105007

concentrations recorded range from very little to complete absence [18]. The low LC50 acute toxicity tests results reflects the toxicity of effluents discharged into water bodies which raises serious concern about the continued introduction of the effluent into the Lagoon ecosystem. The result derived from this study is consistent with the low toxicity values of effluents from paint, textile and chemical as obtained by several author [20]. The induction of micronuclei in erythrocytes of exposed fish in this study agrees with the observation of [17] who reported a significant increase in the frequency of micronucleated erythrocytes of Clarias lazera exposed to textile mill effluent. Similarly, induction of micronuclei in peripheral erythrocytes of Oncorhynchus mykiss exposed to textile industry effluent was shown by [15]. .Molecular and cellular level effects have also been linked with exposure to effluents from industries including textile industries [21] observed that after 15 days exposure to sub-lethal concentrations of the effluent, assessment revealed cellular aberrations (genotoxic effects) which impacts at the cellular and possibly sub cellular levels. This generates serious concerns about the far reaching effects of effluents which are continuously introduced into surrounding water bodies on fishes, shell fishes and other aquatic animals especially edible ones. Micronuclei assay with fish show potential as monitoring techniques for detecting genotoxic substances in water. The decrease in the mitotic index (MI) with increasing concentrations of industrial textile effluents as well as the formation of chromosomal aberrations such as vagrant, sticky chromosomes, binucleus and c-tumors, severe reduction in root length can be attributed to the presence of some cytotoxic and genotoxic compounds in the effluent. Further studies need to be carried out to identify the specific agents responsible for the genotoxic effects. V. CONCLUSION The results obtained from this study indicate the textile effluent being discharged into the Lagos lagoon have

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genotoxic potential and is capable of causing significant ecological disruption in the receiving environment. The increase in the formation of micronuclei and nuclear anomalies in blood erythrocytes of Clarias gariepinus, as well as, chromosomal aberrations such as vagrant, sticky, binucleus and c-tumors in Allium cepa were identified as good genotoxic biomarkers for monitoring impact of industrial effluents in the environment. ACKNOWLEDGEMENTS Authors wish to express their gratitude to Mr F.O Obu of the Department of Cell Biology and Genetics, Mr S.Samuel for analysis of data. REFERENCES
[1] J.C Akan, V. O. Ogugbuaja, F. I. Abdulrahman, and J. T. Ayodele, Pollutant levels in effluent samples from tanneries and textiles of Kano industrial areas, Nigeria, Global Journal of Pure and Applied Science, vol. 15(3), pp. 343-352, 2009. T. Akpata, J.A. Oyenekan, and D.I. Nwankwo, Impact of organic pollution on the bacterial plankton and benthic population of Lagos lagoon, Nigeria, Intl. J. Eco. Env. Sci., vol. 17, pp. 1-10, 1993. N.H. Amaeze, Comparative account of the physiochemical properties; biodiversity and feeding relationships in two creeks in the university of Lagos. Nigeria, M. Sc thesis, University of Lagos, Lagos, Nigeria, 2008. K.R. Carrasco, K.L. Tilbury, and M.S. Mayers, Assessment of the piscine micronuclei test as an in situ biological indicator of chemical contaminants effects, Can. J. Fish Aquat. Sci., vol. 47, pp. 2123-2136, 1990. T. Cavas, and S. Ergene-Gozukara, Micronuclei, nuclear lesions and interphase silver-stained nucleolar organizer (AgNORs) as cytogenotoxiciy indicators in Oreochromis niloticus exposed to textile mill effluent, Mutat. Res., vol. 538, pp. 81-91, 2003. S. Chandra, L.K.S Chauhan, R.C. Murthy, P.N Saxena, P.N Pande, and S.K Gupta, Comparative biomonitoring of leachates from hazardous solid waste of two industries using Alium test, Sci. Total Environ., vol. 347(2), pp. 46-52, 2005. L.C.M Egito, M.G. Medeiros, S.R.B. Medeiros, and L.F. Agnez-Lima, Cytotoxic and genotoxic potential of surface water from the Pitimbu river, Northeastern/RN Brazil, Mol. Biol., vol. 30(2), pp. 435-441, 2007. Fiskesjo, The Allium test as a standard in environmental monitoring, Hereditas, vol. 102, pp. 99 112, 1985. [9]

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W.F. Grant, Chromosome aberration assays in Allium: A report of the U.S Environmental Protection Agency gene-tox program, Mutat. Res., vol. 99(3), pp. 273-291, 1982. J.A. Heddle, M.C. Cimino, M. Hayashi, F. Romagna, M.D. Shelby, J.D. Tucker, P. Vanparys and J.T. MacGregor, Micronuclei as an index of cytogenetic damage: Past, present and future, Environ. Mol. Mutagen., vol. 18, pp. 277-291, 1991. Hoofman, R.N. and W.K. Raat, 1982. Induction of nuclear anomalies (micronuclei) in the peripheral blood erythrocytes of the eastern mudminnow Umbra pigmaea by ethyl methane sulphonate. Mutat. Res., 104: 147-152. W.O Odiete, Enviromental Physiology of Animals and Pollution. Diversified Resources Publishers, Lagos, 1999. W. Jiraungkoorskul, P. Kosai, S. Sahaphong, P. Kirtputra, J. Chawlab, and S. Charucharoen, Evaluation of micronucleus tests sensitivity to freshwater fish species, Res. J. Env. Sci., vol. 1(2), pp. 56-63, 2007. S. Karthikeyan, M. Jambulingam, P. Sivakumar, A.P. Shekhar and J. Krithika, Impact of Textile Effluents on Fresh Water Fish Mastacembelus Armatus, E-Journal of Chemistry, vol. 3(13), pp. 303306, 2006. M.J. Marlasca, C. Sanpera, M.C., Riva, R.Sala, and S. Crespo, Micronucleus test in erythrocytes of Barbus plebejus (Teleostei, Pisces) from two natural environment: a bioassay for the in situ detection of mutagens in freshwater, Mutat. Res., vol. 367, pp. 245251, 1998. M.A. Marin-Morales, and D.M. Leme, Allium cepa test in environmental monitoring: A review on its application, Mutation Research, 682: 7181. 2009. Odeigah, and O. Osanyinpeju, Genotoxic effects of two industrial effluents and ethylmethane sulfonite in Clarias lazera, Food Chem. Tox. Vol. 33, pp. 501 505, 1995. A.A Otitoloju, Joint action toxicity of heavy metals and their bioaccumulation in benthic animals of the Lagos lagoon, Ph.D thesis, University of Lagos, Nigeria, 2000. J. Rank, The method of Allium anaphase-telophase chromosomeaberration assay, Ekologia, vol. 1, pp. 38 42, 2003. Samuel, F.I. Osuala, and P.G.C. Odeigah, Cytogenotoxicity evaluation of two industrial effluents using Allium cepa assay, Afr. J. Biotech., vol. 4(1), pp. 21-27, 2010. M. Sumath, K. Kalaisevi, M. Palanivel, and P. Rajaguru, Genotoxicity of textile dye effluent on fish (Cyprinus carpio) measured using the comet assay, Bul. Env. Contam. Toxico., vol. 66, pp. 407-414, 2001. Zagal, and B. Mazmanci, Oxidative stress response in Nile tilapia (Oreochromis niloticus) exposed to textile mill effluent, Toxico.Ind. Health, vol. 27(1), pp. 81-85, 2011.

[10]

[11]

[12] [13]

[14]

[15]

[2]

[3]

[16]

[4]

[17]

[18]

[5]

[19] [20]

[6]

[21]

[7]

[22]

[8]

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