ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 15,153- 16 I(1 988)

Branchial Pathogenesis in a Freshwater Fish, Puntius conchonius Ham., Chronically Exposed to Sublethal Concentrations of Cadmium

TEJENDRA S. GILL, JAGDISH C. PANT, AND HEMA TEWARI

Department of Zoology, Faculty of Science, Kumaun University, Naini Tal263 002. India

Received April 15. 198 7

Effects of sublethal concentrations, 630 and 840 &liter (0.05 and 0.066 fractions of the 96- hr LC&, of cadmium chloride on the gills of a freshwater fish, Puntitls conchonius, were exam- ined light microscopically during a 12-week exposure. The secondary gill lamellae showed dis- rupted epithelium, necrosis, accumulation of cellular debris, capillary congestion, and wilting of the pillar cell system. Hypertrophy and hyperplasia of chloride cells as well as partial or com- plete fusion of secondary lamellae also occurred in the Cd-exposed fish. Branchial lesions to- gether with coagulation film anoxia are likely to result in serious respiratory distress and related tissue hypoxia. Q 1988 Academic Press, Inc.

INTRODUCTION

Cadmium, the so-called dissipated element (Fulkerson et al., 1973), is extensively used in electroplating and production of energizers, dyes, and plastic stabilizers (Hut- ton, 1983), and has been listed as the second most hazardous metal in the marine environment (Ketchum et al., 1975). During prolonged exposure, Cd is known to accumulate in the kidneys with a slow turnover and the critical concentration in the renal cortex for protection of human health is stated to be 200 pg/g fresh weight (F&erg et al., 1974; WHO Task Group, 1977). In the people occupationally exposed, Cd causes several ailments affecting bones, kidneys, lungs, liver, and testes (Lauwerys et al., 1979; Nomiyama, 1981).

Like many other water-borne pollutants, Cd poisoning in fishes is known to cause both surticial and systemic morphopathological changes of varying severity (Gardner and Yevich, 1970; Hawkins et al., 1980; Stromberg et al., 1983; Oronsaye and Bra- field, 1984). The gills in fishes are likely to be impaired structurally for they offer a large surface area that comes in direct contact with the dissolved toxicant. Therefore, monitoring of branchial pathology under Cd exposure may have considerable diag- nostic value in assessing the adverse effects on the respiratory and extrarenal func- tions normally performed by the fish gills. Previous investigations on the effects of Cd in a common freshwater bony fish, Puntius conchonius, have revealed a variety of biochemical and hemopathological responses (Gill and Pant, 1983, 1985. 1986). This report describes branchial lesions in P. conchonius under chronic exposure to sublethal concentration of cadmium.

MATERIAL AND METHODS

Animals and test procedure. Adult P. conchonius (Order Cypriniformes), average weight 5 g, were hand-netted from the local lake (altitude 1937 m) and transported to the laboratory in plastic buckets. The animals were maintained in a 400-liter stor-

153 0147-65 13/88 $3.00 Copyright Q 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

154 GILL, PANT, AND TEWARI

age tank exposed to natural photopcriod and temperature regimes. Commercial fish food was provided ad libitum, both during acclimatization and during exposure to cadmium. The average physicochemical characteristics ofthe test water were pH 7.3, EDTA hardness 46 mg CaCO&er, dissolved oxygen 8.5 mg/liter, and temperature 11.8”C. The 96-hr LCSO for CdCl2 in this water was determined to be 12.653 mg/liter.

Exposures were carried out in 35-liter glass aquaria stocked with 25 fish per tank. Groups I and II were exposed to 630 and 840 pg CdClJiter (0.05 and 0.066 fractions of the 96-hr L&J, respectively, and monitored for 12 weeks. A tank of control fish, group III, without CdC& was maintained in a similar manner. All tanks were con- stantly aerated and the test medium was renewed every 2 weeks. Six individuals were removed from each of the control and experimental tanks after completion of expo- sures of 4,8, and 12 weeks’ duration.

Samplings and histological techniques. At the time of sampling, fish were quickly decapitated and the gills of both control and Cd-exposed individuals were collected for histological examination. The operculum was lifted and the gill arches were care- fully removed and placed in freshly prepared aqueous Bouin’s fluid for 24 hr. There- after, they were treated with a decalcifier for 4 hr and thoroughly washed in running water. The complete gill arches were dehydrated in an ascending ethanol series, cleared in xylene, and embedded in paraffin blocks. Sections of 4-6 grn were cut on an American Optical microtome, and stained with Delafield’s hematoxylin using eo- sin as counterstain. Gill Iamellae were examined light microscopically for pathologi- cal lesions and photographed with an Olympus PM 6 camera.

RESULTS

The branchial morphology in P. conchonius is comparable to that in other bony fishes. Each gill arch bears two rows of primary gill lamellae (primary filaments) which in turn have a series of flat, thin, leaf-like secondary lamellae on their upper and lower surfaces. Each secondary lamella consists of interconnecting blood spaces supported by pillar cells and their flanges, and covered by an epithelium (Fig. 1). At the bases of secondary Iamellae and in the interlamellar crypts are found the chloride cells and mucocytes.

In P. conchonius exposed to 630 pg CdClJliter, severe branchial lesions were ob- served after 4 weeks. The lesions were diffuse and characterized primarily by a dis- rupted epithelium, coagulation necrosis of epithelial cells, and accumulation of cellu- lar debris in the secondary lamellae. There was capillary congestion and wilting of pillar cell system particularly at the distal ends of secondary lamellae. In addition, hypertrophy and hyperplasia of the epithelial and chloride cells were conspicuous at the bases of secondary lamellae and in the interlamellar crypts (Fig. 2).

A 12-week exposure to 630 pg CdClJliter resulted in gross degeneration and curl- ing of secondary lamellae in the Cd-exposed fish (Fig. 3). Exposure to higher Cd con- centration, 840 fig CdClJiter, for 12 weeks caused lamellar fusion due to simple apposition as well as hypertrophy and hyperplasia of epithelial cells. The fusion OG curred either at the bases or occasionally along the entire length of secondary lamellae (Fig. 4). During Cd exposure, all the fish secreted copious mucus from all over the body surface, and at the time of sacrifice, a film of coagulated mucus was invariably noticed on the gills.

CADMIUM PATHOGENESIS IN Puntius conchonius 155

FIG. 1. Gill lamellae in the control fish. Hematoxylin & eosin.

DISCUSSION

The gills of Cd-exposed fish have been examined by several investigators. Gardner and Yevich (1970) reported pathological changes in the gills of the killifish, Fundulus heteroclitus, exposed to 50 mg Cd/liter sea water, and suggested that death was caused

156 GILL, PANT, AND TEWARI

FIG. 2. Gill lamellae of fish exposed to 630 pg CdQ/liter for 4 weeks. Note disrupted epithelium, col- lapsed pillar cell system (filled arrow), and hyperplasia of chloride cells (blank arrow). Hematoxylin & eosin.

by impairment of respiratory and osmoregulatory functions through reduction in the gill surface. Young fathead minnows, Pimephalespromelas, exposed to 12 mg CdC12/ liter for 96 hr and then moved to clean water and monitored for 30 days, revealed

CADMIUM PATHGGENESIS IN Puntius conchonius 157

FIG. 3. Gill filaments of fish exposed to 630 rg CdCI,/liter for 12 weeks showing grossly curled secondary lamellae. Hematoxylin & eosin.

epithelial necrosis characterized by nuclear pycnosis, karyorhexis, cellular sloughing, and eosinophilic debris (Stromberg et al., 1983). In the present study, severe branchial lesions were seen in the Cd-exposed P. conchonius. The first signs of externally in- duced gill damage included lifting of lamellar epithelium, necrosis, and accumulation

158 GILL, PANT, AND TEWARI

FIG. 4. Gill lamellae of fish exposed to 840 pg CdClJliter for 12 weeks. Note partly (filled arrow) and completely fused lamellae (blank arrow) with hypertrophied epithelium. Hematoxylin & eosin.

of cellular debris. Due to the corrosive action of Cd, the affected gill lamellae showed a collapsed pillar cell system and capillary congestion. Partial or complete fusion of lamellae also occurred in the Cd-exposed P. corzchonius. These necrotic changes were accompanied by a diffuse hypertrophy and hyperplasia of chloride cells especially at

CADMIUM PATHOGENESIS IN Puntius conchonius 159

the bases of secondary lamellae. This obviously occluded the interlamellar spaces through which the respiratory current normally flows.

Changes in the distributional pattern as well as a numerical increase in the chloride cells have been described. Exposure to 0.5 to 1.5 mg Cd/liter in both soft and hard water (hardness 132 and 299 mg/liter as CaCO3, respectively), caused an initial in- crease followed by a pronounced decline in the number of chloride cells in the stickle- back, Gasterosteus aculeatus (Oronsaye and Brafield, 1984). The authors opined that the chloride cell hyperplasia occurs in response to the need to eject the Cd ions ab- sorbed by the gills. The chloride cell hypertrophy and hyperplasia in P. conchonius occurs as a natural protective response but it results inadvertently in a decreased lamellar surface which apparently disrupts respiratory function culminating in hypo- xemia. The respiratory distress is further compounded due to decreased hemoglobin, hematocrit, and erythrocyte counts in the Cd-exposed P. conchonius, which in turn contribute to a reduced oxyphoretic capacity of the blood (Gill and Pant, 1985).

The uptake and toxicity of heavy metals depend considerably on gill epithelial permeability and mucus secretion. Among the factors critical for gill permeability in the freshwater fishes are the environmental calcium and internal levels of prolactin (Hunn, 1985). The Ca uptake from the medium depends on the presence of Ca*+- binding protein, calmodulin, and certain enzymes in the gills. One such enzyme, the calmodulin-dependent Ca’+-Mg ‘+-ATPase, is reported to be directly involved in the Ca” transport across the branchial epithelium in freshwater-adapted American eel, Ang-uilla r&rata (Flik et al., 1984). Under exposure to Cd, the substitution of Ca” by Cd ions occurs in the calmodulin (Suzuki et al., 1985) which may disturb the calmodulin-dependent cellular processes and enzyme activities (Cheung, 1980; Su- zuki et al., 1985). Since, cellular Ca fluxes are calmodulin mediated (Vinci& and Hinds, 1980), changes in the physiological functions of this vital protein may lead to irreversible cell damage (Schanne et al., 1979).

Ameliorative effects of Ca in Cd-induced toxicity in cultured rat hepatocytes have been demonstrated (Sorensen et al., 1984). It is suggested that because of similar ionic radii of Ca and Cd (0.99 and 0.97 A, respectively), the Cd replaces Ca on the phospholipid headgroups of plasma membranes (Schaltz and Marinetti, 1972). Pres- ence of sufficient Ca would, however, impede the Cd influx. Cd binding to plasma membranes might represent the first step in the expression of Cd-induced cytotoxicity (Sorensen et al., 1984).

It seems likely that pathological changes observed in the gills of P. conchonius result from ( 1) Cd-induced alterations in the cellular Ca fluxes, and/or (2) a direct cytotoxic action of Cd on the epithelial cells. However, it is worth mentioning that although the gills as well as the liver synthesize metallothionein in an endeavor to sequester the Cd, e.g., in Anguilla anguilla (Noel-Lambot et al., 1978), a prolonged exposure to this noxious metal possibly overburdens the detoxifying mechanisms and the dele- terious effects become manifest in the intoxicated fish. Furthermore, the mucus, de- spite being an effective complexing agent (Varanasi and Markay, 1978), affords only limited protection to the fish under heavy metal poisoning. Toxic metal ions like Hg and Cd are absorbed via gills but in the presence of mucus their diffusion is reduced by 50% (Part and Lock, 1983) while it remains unaltered in Ca ions. Accordingly,

the net influx of toxic ions would vary with the duration and intensity of exposure. Present findings in P. conchonius suggest that the severity of branchial lesions is

160 GILL, PANT, AND TEWARI

affected more by the duration of exposure rather than the Cd concentration tested in this study.

CONCLUSIONS

In conclusion, it is suggested that the observed branchial lesions represent early warning signs of the death of cellular constituents of secondary lamellae which fail to tolerate sublethal accumulations of Cd. Collateral examination of kidneys in the Cd- exposed P. conchonius has revealed unequivocal degenerative changes mainly in the proximal segments of renal tubules, while distal segments and collecting tubules and ducts remain unaffected (Gill et al., unpublished observations). Therefore, it appears that the Cd-mediated sublethal responses are not confined to the surficial organs be- cause the metal ions find their way into systemic organs as well. Nevertheless, moni- toring of branchial pathology may help elucidate the threshold concentration of Cd which the fish can tolerate without obvious adverse effects on health. Further, com- pensatory changes in respiration, ionoregulation, and cardiovascular functions, be- sides the metal sequestration by metallothionein, are possibly some of the reasons for the experimental fish surviving the stipulated 1Zweek exposure period in the pres- ent study.

ACKNOWLEDGMENTS

Financial support for this project was received from the Council of Scientific and Industrial Research, New Delhi, and the University Grants Commission, New Delhi. Thanks are due to the Department of Zoology for the laboratory facilities.

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