Introduction

Tilapia mossambica is a commercial teleost fish, which are widely cultured for human consumption¹. Dyes are used in many industries to colour their products such as textile, rubber, paper, plastics, leather, cosmetics, food, paint and mineral processing industries. Coimbatore is an important industrial city of Southern part of India having more than 30,000 small, medium and large industries including textile dyeing units. The dyeing units discharge their effluents into prevailing drainage and ultimately reach lakes, wetlands and River Noyyal in the city². Massive death of fish in Puttu vikki dam near dyeing units in Coimbatore was reported in the news paper, Dina Thanthi³. Dye bath effluent, in particular are not only aesthetic pollutants by nature of their colour, but also can interfere light penetration through water bodies⁴ and thus affects the aquatic organisms.

Acid blue 113, a dark blue powder is extensively used as textile dye having diazo group within the molecular structure⁵. Azo dyes leads to formation of aromatic amines and these are known mutagens and carcinogens to human beings⁶. Dyes have been reported to precipitate in aquatic organisms leading to hematological and histopathological alterations in vital organs⁷. Biochemical changes in blood and tissue of fish under exposure to toxicant may be used to predict the toxic effects⁸.

Various reports are available on the toxic effects of acid blue dyes such as acute toxicity bioassay in fish⁹, allergen susceptibility and skin sensitization in fish, rat and rabbit^10,11, colour change in internal organs in fish and rat¹², changes in mineral constituents in fish liver and kidney¹³. The biochemical effects of acid blue -113 in various organs of fish need attention. In this context acute toxic effect of acid blue – 113 on the biochemical parameters of the fresh water fish Tilapia mossambica is attempted.

Materials and Methods

Test animal

Tilapia mossambica of both sexes (with size i.e. length = 8 to 13cm and weight = 15 to 20g) were procured from Tamil Nadu Fisheries Department, Aliyar Dam hatchery at Coimbatore district, and brought to the laboratory in oxygen packs. Fish were then transferred into glass aquaria filled with aerated (chlorine free) tap water and stock fish were provided with commercial feed at the rate of 1% live weight per day. They were acclimatized for 15 days in the laboratory. Fish were not fed for 24 hours prior to testing, nor for the duration of the tests.

Test material

The textile dye, Acid blue 113 a diazo dye was purchased from the textile chemical shop in Coimbatore, Tamil Nadu.

Acute toxicity bioassay test (LC₅₀ 96 hours)

Test was designed according to the static bioassay procedure of Standard Methods for the Examination of Water and Wastewater¹⁴. The quality of experimental dilution water was analysed according to Standard Methods¹⁴. Test concentrations were 5, 10, 20mg/l. Experimental troughs of 60 liters capacity was used for each exposure and 10 fish were introduced in each trough. Duration of all tests was 96 hours and fish were observed at 24 hour intervals to note the mortality in each trough. As a control, 10 fish were maintained throughout the test period under identical conditions. The LC₅₀ value was derived by plotting the data on semi logarithmic coordinate paper.

Experimental design

There were five groups (4 troughs with sublethal doses and another trough with control) and each group has 10 fish. One tenth of lethal concentration was considered for 24, 48, 72 and 96 hours for test exposure.

Sampling

After the exposure periods, sublethal and control group fish were sacrificed for estimation of biochemical parameters. For the analysis of biochemical parameters, liver and muscle tissues were dissected out from the control and experimental fish. The tissues were weighed and centrifuged at 4,000 rpm using double distilled water. The supernatant was stored at 2⁰C for analytical purpose. Colorimetric determination was performed using BTR – BIOTRONE 810(German) semi auto analyser.

 Statistical analysis

All experiments were repeated for three times and values are expressed as mean ± Standard deviation. The individual values of all parameters were compared by Student’s t – test (5% significance) and also percentage of decrease over control was noted.

Results and Discission

Biochemicals such as carbohydrate, protein and lipid and enzymes such as Glutamate Oxaloacetate Transaminase (GOT) and Glutamate Pyruvate Transaminase (GPT) were analysed in liver and muscle to determine the toxic effects of textile dye Acid blue – 113 in Tilapia mossambica. The biochemical aspects of liver are tabulated in Table 1 and muscle in Table 2. The study showed decrease in carbohydrate, protein and lipid content in both liver and muscle of dye exposed fish when compared to fish under control. The enzymatic aspects of liver are shown in Table 3 and for muscle in Table 4. GOT and GPT values in the liver and muscle are higher in experimental fish than in control.

Table 1: Biochemical Parameters In Liver Of Tilapia Mossambica On Exposure To Acid Blue – 113.

Table 2: Biochemical Parameters In Muscle Of Tilapia Mossambica On Exposure To Acid Blue – 113

*Significant at 5% level,   SD = Standard Deviation, -indicates decrease over control.

Table 3: Enzyme Parameters In Liver Of Tilapia Mossambica On Exposure To Acid Blue – 113

*Significant at 5% level,   SD = Standard Deviation, + indicates increase over control.

Table 4: Enzyme Parameters In Muscle Of Tilapia Mossambica On Exposure To Acid Blue – 113.

*Significant at 5% level,   SD = Standard Deviation, + indicates increase over control.

Carbohydrates are essential components of all living organisms and are, in fact, the abundant class of biological molecules. In the present investigation, decline in the carbohydrate content of liver and muscle in all the experimental fish shows the primary metabolic symptom that can be noticed in organisms subjected to stressful situations. The significant decrease (P<0.05) of carbohydrates in the tissues indicated the excess utilization of glucose and the reserve polysaccharide glycogen to withstand the dye induced toxicities. In conformity with this finding, previous reports on Tilapia zilii to polluted water¹⁵; Ophiocephalus to dairy effluent¹⁶; Catla catla to cadmium chloride¹⁷ and Catla catla to mercury chloride¹⁸ are available.

Proteins are highly sensitive to pollutants and are one of the earliest indicators of toxic effects¹⁹. It may be influenced by a large number of exogenous substances, mainly through a reduction of protein synthesizing capacity of endoplasmic reticulum in the cells. The results of the present study showed significant decrease (P<0.05) in protein content that might be attributed to diversification of energy to meet the impeding energy demands when the fish is under stress or altered enzyme activities. Decrease of protein in fishes due to toxicants was reported in Rainbow trout to malachite green²⁰; in Catla catla to cadmium chloride¹⁷; in Catla catla to mercuric chloride¹⁸.

Lipids are storage form of energy. The present study showed decreased level of lipid in muscle and liver of fish exposed to Acid blue – 113 when compared to control. The reduction in lipid was resulted due to their utilization under stress¹⁶. In conformity with the present findings, previous reports on Cyprinus carpio to tannery effluents²¹; Ophiocephalus to dairy effluents¹⁶  and Catla catla to cadmium chloride¹⁷ are available.

Decrease or increase in enzyme activity represents the response of organism to any kind of stress. GOT and GPT are two key enzymes known for their role in the utilization of protein and carbohydrates. Increased GOT and GPT activity in liver and muscle of the experimental fish exposed to Acid blue – 113 was noticed. Similar findings of increased transaminase activity on exposure to dye stuffs were reported in Clarias lazera²². Increased transaminase activity of the fish was due to stress caused by toxicity.

The results indicate that the dye induced, stressful environment to the fish. The organs considered for the study i.e., liver and muscle of fish showed remarkable changes to exposure of the dye. Further studies on the effects of chronic exposure of the dye on the other organs can reveal the basic understanding of the toxic effects.

 Acknowledgements

The authors are thankful to Dr. S. Ravisankar, Associate Professor of Zoology, Kongunadu Arts & Science College, Coimbatore, Tamil Nadu for the valuable guidance and assistance in preparation of the manuscript and to Rev.Fr.G.P.Mathen, Lecturer, St. Stephen’s College, Pathanapuram, Kerala for laboratory facilities and technical support.

References

1. Wardhaugh, A.A., Dominent lethal mutation in T.mossambica (Peters) elicited by Myleran. Mutat. Res., 88, 191 – 196, (1981).
2. Mohanraj, R., Sathish Kumar, M., Azeez, P.A. and Sivakumar, R., Pollution status of wetland in urban Coimbatore, Tamil Nadu, India. Bull. of Environ. Contam. Toxicol., 64, 638 – 643, (2000).
3. Dina Thanthi (Tamil News Paper), Massive death of fish in Puttu vikki dam. 12^th June (2008).
4. Yasin Binti Che Ani, Adsorption studies of dyes using clay based and activated carbon adsorbents. M.Sc. Thesis, School of Chemical engineering, University Sains Malaysia. (2004).
5. Tae – Kung Kim, Mee – Kyung Kim, Yong – Jim Lim and Young A S., Degradation of the disazo acid dye by the sulphur containing amino acids of wool fibers. Dyes & Pigments. 67, 127 – 132, (2005).
6. Brown, M.A., De Vito, S.C., Predicting azo dye toxicity. Cri. Rev. Env. Sci. Tec., 23, 249 –324, (1993).
7. Afaq, S. and Rana, K.S., Impact of leather dye on total protein of fresh water teleost, Cirrhinus mrigala(Ham.). Asian J. Exp. Sci., 23, 299-302, (2009).
8. Ramesh, M. and Saravanan, M., Haematological and Biochemical responses in a fresh water fish Cyprinus carpio exposed to Chlorpyrifos. Inter. J. Integra. Biol., 3, 80 – 83, (2008).
9. Linda, W. L., and James, C. L. III., Acute toxicity of 46 selected dyes to the fathead minnow, Pimephales promelas. UNC Waste water research center, Department of Environmental Sciences and Engineering, School of Public Health, University of  NorthCarolina 27514 for Ecology Committee American Dye Manufacturers Institute, INC. (1972).
10. Anonymous, Orange textile dye. National Industrial Chemical notification and assessment scheme (NICNAS). File No: STD/1008, In: National Occupational Health and Safety Commission, Australia.(2003).
11. Anonymous, Acid blue113.21cclab.com. (2008).
12. SCCP(Scintific Committee of Consumer Products)., Acid Blue 62 (COLIPA1 n° C 67). European Commission, Health & Consumer Protection Directorate General, Directorate C – Public Health & Risk Assesssment. (2005).  http://pharmacos.eudra.org/F3/cosmetic/doc/HairDyeStrategyInternet.pdf.
13. Karthikeyan, S., Jambulingam, M., Sivakumar, P., Shekhar, A.P. and Krithika,J., Impact of textile effluents on fresh water fish Mastacembelus armatus (CUV & Val). E – Journal of  Chemistry, 3, 303 – 306, (2006).
14. APHA, Standard methods for the examination of water and waste water, 21^st Edn., Washington DC, USA. (2005).
15. Abdel – meguid, N., Kheirallah A.M., Abou-Shabana, Adham K. and Abdel- Moneim A., Histochemical and Biochemical changes in Liver of Tilapia Zilli G as a consequence of water pollution. Online journal of Biological sciences., 2, 224-229, (2002).
16. Sabarinathan, D., Indra, V., Vanitha, A. and Darwin, S., Effect of dairy effluent on the survival, protein, carbohydrate and fat metabolism of fresh water fish Ophiocephalus. Poll. Res., 25, 675 – 676, (2006).
17. Sobha, K., Poornima, A., Harini, P., Veeraiah, K., A study on Biochemical changes in the fresh water Fish, Catla catla (Hamilton) Exposed to the Heavy Metal Toxicant Cadmium Chloride. Kathmandu University Journal of Science, Engineering and Technology,1, 1-11, (2007).
18. Martin Devaprasath, P. and Arivoli, S., Biochemical study of freshwater fish Catla catla with reference to mercury chloride. Iran J. Environ. Health. Sci. Eng. 5, 109 – 116, (2008).
19. Jacob, J.M., Carmichael, N. and Cavanagh, J.P., Ultra structural changes in the nervous system of rabbits poisoned with methyl mercury. Toxicol. Appl. Pharmacol., 39, 249 – 261, (1977).
20. Muhammed A., The effects of a disinfectant (Malachite Green) on blood biochemistry of Rainbow Trout (Oncorhynchus mykiss). J. Fish. Aqua. Sci., 2, 82 – 85, (2007).
21. Ambrose, T., Arunkumar, C.L., Vincent, S. and Lambert, R., Biochemical responses of Cyprinus carpio Var. communis to toxicity of tannery effluent . J. Ecobiol., 6, 213 – 216, (1994).
22. Abdel Moneim, A.M., N.M.Abou Shabana, S.E.Khadre and H.H.Abdel Kader: Physiological and histopathological effects in Catfish (Clarias lazera) exposed to dyestuff and chemical wastewater. Int. J. Zool. Res., 4, 189 – 202, (2008).