Journal of Environmental Biology April 2006, 27(2) 211-215 (2006) ©Triveni Enterprises, Lucknow (India) For personal use only Free paper downloaded from: www.jeb.co.in Commercial distribution of this copy is illegal

Effects of sub-lethal concentrations of zinc on histological changes and bioaccumulation of zinc by kidney of fish Channa punctatus (Bloch)

Pallavi Gupta and Neera Srivastava Fish Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur-302 004, India

(Received: 24 April, 2004 ; Accepted: 25 October, 2004)

Abstract: Fresh water fish , Channa punctatus were exposed to three sub-lethal concentrations of zinc (10 mg/l, 15 mg/l and 25 mg/l) for 15 days. The effects of this exposure have been studied on bioaccumulation of zinc and histology of the kidney at intervals of 8, 10 and 15 days. Statistically significant increase in zinc concentration was noted in fish of all treated groups. Simultaneously, severe histological changes were noted in the kidney of all exposed groups. Both, bioaccumulation and histopathological changes were dose and duration dependent. Post exposure recovery in fish was noted after transferring these fish to normal tap water for another fortnight. Slow elimination of zinc was observed though the concentration of zinc remained significantly higher than controls till the end of the experiment. Histopathological changes observed in the kidney also persisted. However, by the termination of the experiment kidney of group II showed signs of mild recovery.

Key words: Channa punctatus, Zinc, Kidney, Histology, Bioaccumulation.

Introduction

Untreated community wastes, use of fertilizers and pesticides as well as dumping of organic and inorganic wastes from industries are increasing environmental pollution to a great extent. Heavy metals have been recognized as strong biological poisons because of their persistent nature, toxicity, tendency to accumulate in organisms and undergo food chain amplification (Kamble and Muley, 2000; Dinodia et al., 2002); they also damage the aquatic fauna including fish.

Among heavy metals, zinc is used in various forms which eventually finds its way into the river or sea. Excessive zinc enters the environment as a result of human activities such as mining, purification of zinc, lead and cadmium ores, burning of coal and burning of waste. Although, small quantities of zinc are required for the normal development and metabolism (Gupta and Sharma, 1994; Srivastava and Sharma, 1996; Srivastava and Kaushik, 2001; Shukla et al., 2002), but if its level exceeds the physiological requirements, it can act as a toxicant. This results in general enfeeblement, retardation of growth and may bring about metabolic and pathological changes in various organs in fishes (Sharma and Sharma, 1994; Singh and Gaur, 1997).

Since fish population is an important component of the food chain any effect of such pollution would in the due course, have adverse influence on the nutritive value of fish and on man through their consumption.

Some histopathological works are available on the effects of different pollutants on fish kidneys (Banerjee and Bhattacharya, 1994; Dhanapakiam and Premlatha, 1994) but much is not known about the effect of zinc on the histopathology of exposed fishes. Accumulation of zinc in various organs of fish has been described by a few workers (Handy and Eddy, 1990; Gupta and Sharma, 1994; Pandey et al., 1995; Singh and Gaur, 1997).Results of above mentioned

studies are contradictory and has not been correlated with the extent of histopathological changes encountered in the tissues.

Keeping the above facts in mind the present study was designed to observe the toxic effects of zinc on a freshwater edible fish, C. punctatus. The study of histopathological changes in the kidney has been correlated with the uptake, retention and release of zinc by this organ on exposure to zinc and after withdrawal of treatment.

Materials and Methods

Live specimen of freshwater fish, C. punctatus were collected from local water bodies and acclimatized for two weeks under laboratory conditions. The physico-chemical characteristics of water were analysed as per methods given in APHA et al. (1989) (Table 1). On the basis of 96 hr LC50 of zinc for C. punctatus three sub-lethal concentrations i.e. 10 mg/l, 15 mg/l and 25 mg/l were selected for the present study. Fish were exposed to these concentrations of zinc for 15 days. LC50 value was calculated by developing a regression equation described by Snedecor and Cochran (1967). Zinc was given in the form of zinc sulphate dissolved in distilled water. Group I served as control. Observations were made on 8th, 10th and 15th day. After 15 days these fish were transferred to normal tap water and recovery responses were observed for 15 more days. Group I served as control. Recovery responses were observed on 8th, 10th and 15th day.

At the end of specified periods of experimental exposure, the kidney tissue was weighed as 30 mg for each sample and digested in a diacidic solution (HNO3 : HClO4 = 5:1). Digested samples were analyzed by atomic absorption spectrophotometer in air acetylene flame at 213 µ for estimation of the zinc content. Data obtained was subjected to statistical analysis. The difference between control group and experimental groups were analysed with the help of student ‘t’

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Table – 1: Physico-chemical characteristics of water.

Temperature 25ºC to 27 ºC

pH 7-7.2 dissolved oxygen 7-7.5 mg/l Hardness 210-215 mg/l Chloride content 35-38 mg/l Alkalinity 67-69 mg.\/l

test. F-test or ANOVA i.e. "Analysis of Variance" was used to evaluate the differences amongst various experimental groups. For histopathological studies routine histological procedure was followed. Sections were cut at 5 µ and stained with Harris’ haematoxylin and eosin.

Results and Discussion

In the present study zinc concentration in kidney of treated groups showed statistically significant increase (p< 0.01) in all groups and at all intervals in comparison to control (Table 2). Zinc concentration in treated groups increased with an increase in dose and duration of treatment. Statistical analysis by ANOVA also shows that uptake of zinc is significantly (p< 0.01) influenced both by dose and duration of the experiment. Similar to the present study high accumulation of various metals has been noted in the kidney of fish by Kumada et al. (1973), Dallinger et al. (1987), Mohammed Al-Mohanna (1994), Gupta and Sharma (1994) and Thiruvalluvan et al. (1997). Dallinger et al. (1987) recorded a high accumulation of several heavy metals in liver and kidney and suggested that these organs are target organs for final deposition of various heavy metals.

High zinc concentration in the kidney of treated fish is probably a result of the kidney being one of the major organs for detoxification and elimination of metallic pollutants (Dallinger et al., 1987). The dose levels of zinc administerated in the present experiment appear to affect the detoxication mechanism within the kidney thereby retarding metal elimination and enhancing its accumulation.

Results of recovery phase still show highly significant (p< 0.01) retention of zinc by the kidneys of C. punctatus in comparison to control (Table 3). ANOVA also show that the variation between treated groups and between intervals are highly significant (p< 0.01). However, a mild trend towards elimination of zinc could be observed in all post exposure recovery groups with the passage of time. Norrgren et al. (1991) have reported a decrease in aluminium of the kidney in Phoxinus phoxinus during recovery period at pH-7 in aluminuim free water after pre-exposure to aluminium in an acidic medium for 12 days. Gupta and Sharma (1994) observed high retention and slow elimination of zinc by the kidney of fingerlings of Cirrihinus mrigala maintained in normal freshwater for a period of 96 hr after pre-exposure to zinc for 96 hr.

Slow elimination of zinc in the present study indicates that the stress of pre-exposure probably alters certain basic biodegradation mechanisms and that a longer period may be required for zinc to fall within normal levels. Thus accumulation of zinc is much quicker than its elimination as retention of zinc could be noted upto 15 days.

Histopathological changes in the kidney were observed at day 8 and continued till the termination of the experiment. Renal tubules became highly expanded, their epithelial lining was distinctly separated from the tubular cells (Fig 2). Some renal tubules were characterized by loss of cellular integrity. Dilation, oedema and hypertrophied nuclei of renal tubules are also noted (Fig. 3). Glomeruli show vacuolization and disorganized blood capillaries (Fig. 4). Necrosis and pyknotic nuclei can be observed in mesenchymal tissue (Fig. 3). Damage becomes more pronounced by day 15 and is more severe in groups III and IV.

Pathological changes have earlier been reported in the kidney of fishes exposed to various pollutants (Banerjee and Bhatacharya, 1994; Anitha Kumari and Sree Ram Kumar, 1997).

Pathological changes, observed in the present study are severe enough to cause impairment in the functioning of the

Table – 2: Zinc accumulation by the kidney (µg/g) of fish C. punctatus subsequent to exposed to sub-lethal concentrations of zinc.

Day 8 Day 10 Day 15

Group 1 (Control) 108.88 ± 1.67 107.94 ± 1.69 110.10 ± 1.30 Group II (10 mg/l) 207.66 ± 0.98** 225.77 ± 0.56** 269.88 ± 0.88** Group III (15 mg/l) 252.23 ± 0.97** 264 ± 1.05** 293.33 ± 1.01** Group IV (25 mg/l) 316.94 ± 0.96** 328.16 ± 1.43** 359.75 ± 1.89**

**Significant at p< 0.01

Table – 3: Zinc concentration in the kidney (µg/g) of fish C. punctatus pre-exposed to sub-lethal concentrations of zinc.

Day 8 Day 10 Day 15

Group 1 (Control) 105.16 ± 1.63 109.32 ± 0.91 102. ± 1.12 Group II (10 mg/l) 245.83 ± 0.93** 235.38 ± 0.73** 219.55 ± 0.75** Group III (15 mg/l) 273.55 ± 0.80** 254.10 ± 0.87** 236.38 ± 0.83** Group IV (25 mg/l) 332.99 ± 0.91** 297.27 ± 0.72** 268.49 ± 0.71**

**Significant at p<0.01

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Fig. 1: Photomicrograph of the kidney of Channa punctatus showing normal kidney structure well formed renal tubules (rt), glomerulus (gl) and mesenchyma (ms). X 200.

Fig. 3: Photomicrograph of the kidney of Channa punctatus showing loss of cellular integrity of tubular cells (arrow), oedema (oe) and dilation (dl) of renal tubules. Note necrosis (ne) and pyknotic nuclei (py)in the mesencymal tissue. X 200.

Fig. 5: Photomicrograph of the kidney of Channa punctatus after 15 days recovery period (group II) showing signs of recovery. Note well formed renal tubules (rt) and glomerulus (gl). X 200.

Fig. 2: Photomicrograph of the kidney of Channa punctatus showing enlargement of renal tubules (arrow), desequamation of epithelial lining (ds). Note hypertrophied nuclei (nu) in renal cells. X 200.

Fig. 4: Photomicrograph of the kidney of Channa punctatus showing vacuolization (vc) and disorganized blood capillaries (arrow) in glomerulus. X 200.

Fig. 6: Photomicrograph of the kidney of Channa punctatus after 15 days recovery period (group III) showing still present histopathological damages. X 200.

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kidney. These degenerative changes may be due to altered metabolic activity or due to metal ion-renal tissue interaction as suggested by Gupta and Rajbanshi (1979, 1982) and Sharma and Sharma (1994). These interactions may in turn lead to excretory disorders and interrenal exhaustion (Rasquin and Rosenbloom, 1954). It can also be suggested that damage of nephrons may result in impaired osmotic and ionic regulation since the renal tubular epithelium has a major function in excretion of divalent ions. Singhal and Jain (1997) also subscribe to this opinion.

It has also been observed that if the concentration of heavy metals is very high in the tissue, it may cause severe structural damage (Venkataramana and Radhakrishnaiah, 1987; Anitha Kumari and Sree Ram Kumar, 1997). In C. punctatus histopathological changes in the kidney are accompanied by high zinc accumulation.

Histopathological changes caused by zinc still persist in all pre-exposed groups. However, at the termination of the experiment kidney structure shows clear signs of recovery in group II (Fig. 5). No signs of recovery can be noted in groups III and IV by day 15 (Fig.6). A study conducted by Kaushik and Srivastava (2003) supports the above observations, where the liver of C. punctatus showed mild recovery in histological damage of groups pre-exposed to low concentration of zinc.

Recovery of histopathological condition is inversely proportional to the exposure dose level. Preferential accumulation of metals (Anitha Kumari and Sree Ram Kumar, 1997) is probable cause of histopathological damage of tissue. Similarly in the present study histological damage can be correlated with high retention of zinc in the kidney of groups III and IV. Kidney is involved with detoxification and excretion of pollutants. High accumulation of zinc by kidney suggests that zinc treatment probably dysfunction the detoxification mechanism of this organ and enhance its accumulation and cause histological changes.

Above study suggest that exposure of fish to different concentrations of zinc (10-25 mg/l) for just a fortnight, poses great stress on the fish and elicits severe changes in their histology. The study also indicates that after withdrawal of zinc, recovery is not spontaneous, it very slow but progressive. It can be inferred that fishes are unable to overcome the stress of per-treatment within 15 days, and duration longer than 15 days is required for normalizing the tissue damage and elimination of accumulated zinc.

References

Anitha Kumari, S. and N. Sree Ram Kumar: Histopathological alterations induced by aquatic pollutants in Channa punctatus from Hussain Sagar lake (A.P.). J. Environ. Biol., 18(1), 11-16 (1997).

APHA, AWWA and WPCF: In: Standard method for the examination of water and waste water. (Eds: M.J. Taras, A.E. Greenberg, R.D. Hoak and M.C. Rand), 7th Edn. Am. Publ. Health Assoc. Washington D.C(1989).

Banerjee, S. and S. Bhattacharya: Histopathology of kidney of Channa punctatus exposed to chronic non-lethal levels of elsan, mercury

and ammonia. Ecotoxicol. Environ. Saf., 29(3), 265-275 (1994). Dallinger, R., F. Prosi, H. Segner and H. Back: Contaminated food and

uptake of heavy metals by fish : A review and a proposal for further research. Oceologia (Berlin)., 73, 91-98 (1987).

Dhanapakiam, P. and J. Premlatha: Histopathological changes in the kidney of Cyprinus carpio exposed to malathion and sevin. J. Environ. Biol., 15(4), 283-287 (1994).

Dinodia, G. S., R. K. Gupta and K. L. Jain: Effect of cadmium toxicity on liver glycogen in some fresh water fishes. Proc. XI Natl., Symp. Environ. 236-238 (2002).

Gupta, A.K. and S.K. Sharma: Bioaccumulation of zinc in Cirrihinus mrigala (Hamilton) fingerlings during short-term static bioassay. J. Environ. Biol., 15(3), 231-237(1994).

Gupta, A.K. and V. K. Rajbanshi: Histopathological changes resulting from bioassay copper to Heteropneustes fossilis (Bloch). Proc. Symp. Environ. Biol. 167-172 (1979).

Gupta, A.K. and V.K. Rajbanshi: Cytopathological studies resulting in cadmium bioassay with Heteropneustes fossilis (Bloch). Acta. Hydrochem.Hydrobiol., 10, 345-351(1982).

Handy, R.D. and F. B. Eddy: Influence of starvation on water borne zinc accumulation by rainbow trout, Salmo gairdneri,at the onset of episodic exposure in natural soft water. Water Res., 24(4), 521-527 (1990).

Kamble, G.B. and D.V. Muley: Effect of acute exposure of endosulfan and chlorpyriphos on the biochemical composition of the freshwater fish, Sarotherodon mossambicus. Indian J. Environ. Sci., 4(1), 97-102 (2000).

Kaushik, N. and N. Srivastava: Recovery in zinc induced hepatotoxicity of Channa punctatus. J. Ecophysiol. Occup. Hlth., 3, 199-204 (2003).

Kumada, H., S. Kimura, M. Yokote and Y. Matida: Acute and chronic toxicity, uptake and retention of cadmium in fresh water organisms. Bull. Fresh wat. Fish. Res. Lab. Tokyo, 22, 157-165 (1973).

Mohammed M. Al–Mohanna: Residues of some heavy metals in fishes collected from (Red Sea coast) Jizan, Saudi Arabia. J. Environ. Biol., 15(2), 149-157 (1994).

Norrgren, L., A. Wicklund Glynn and O. Malmborg: Accumulation and effects of aluminium in the minnow (Phoxinus phoxinus L.) at different pH levels. J. Fish. Biol., 39, 833-847 (1991).

Pandey, B.K., U.K. Sarkar, M.L. Bhowmik and S.D. Tripathi: Accumulation of heavy metals in soil,water, aquatic weed and fish samples of sewage-fed ponds. J. Environ. Biol., 16(2), 97-103 (1995).

Rasquin, P. and L. Rosenbloom: Endocrine imbalance and tissue hyperlesia in teleost, maintained in darkness. Bull. Amer. Mus. Water Hist., 104, 359-426 (1954).

Sharma, A. and M.S. Sharma: Toxic effect of zinc smelter effluent to some developmental stages of fresh water fish, Cyprinus carpio (Linnaeus). J. Environ. Biol., 15(3), 221-229 (1994).

Shukla, V., P. Rathi and K.V. Sastry: Effect of cadmium individually and in combination with other metals on the nutritive value of fresh water fish, Channa punctatus. J. Environ. Biol., 23(2), 105-110 (2002).

Singh, M. and K.K. Gaur: Effects of mercury, zinc and cadmium on the proteinic value and their accumulation in trunk muscle of Channa punctatus (Bloch.). Advances Bios., 16(11), 109-114 (1997).

Singhal, R.N. and M. Jain: Cadmium–induced changes in the histology of Kidneys of common carp, Cyprinus carpio (Cyprinidae). Bull. Environ. Contam. Toxicol., 58, 456-461 (1997).

Sndecor, G.W. and W.G. Cochran: Statistical methods, lowa State, University Press, Ames, lowa (1967).

Toxicity of zinc on kidney of fish 215

Srivastava, N. and R. Sharma: Toxicity of zinc in fish (Channa punctatus Bloch.) as influenced by temperature and pH of water. Indian J. Anim. Nutr., 13(2), 87-90 (1996).

Srivastava, N. and N. Kaushik: Use of fish as bioindicator of aquatic pollution. In: Abstracts presented at international congress of chemistry and environment. 16th - 18th Dec. 2001, Indore, India (2001).

Thiruvalluvan, M., N. Nagendran and A. Charles Mahoharan: Bioaccumulation of cadmium and methyl parathion in Cyprinus carpio var. communis (Linn). J. Environ. Pollut., 4(3), 221-224 (1997).

Venkataramana, P. and K. Radhakrishnaiah: Lethal and sub-lethal effects of copper on the protein metabolism of the fresh water fish Labeo rohita (Ham.). Trends Life. Sci., 2(2), 82-86 (1987).

Correspondence to : Dr. Pallavi Gupta D-23, Jagan Path, Chomu House C- Scheme, Jaipur (Rajasthan) India E-mail: pallavi_gupta2006@rediffmail.com Tel.: +91-141-2360667