Summary of Project Work

 The present project was conducted to study the tissue-specific metal bioaccumulation patterns and differences in sensitivity of three fish species viz. Catla catla, Labeo rohita and Cirrhina mrigala  to water-borne and dietary metals. During first year, the acute toxicity of water-borne and dietary metals to three fish species viz. Catla catla, Labeo rohita and Cirrhina mrigala were determined. Three fish age groups viz. 60, 90 and 120-day were tested for acute toxicity (96-hr LC50, LD50 and lethal concentration) of copper, cadmium, zinc, nickel, cobalt and their mixture, separately at constant hardness (200 mgL-1), pH (7) and water temperature (30oC). Three replications, for each test dose, were used for each age and fish species to determine their 96-hr LC50, LD50 and lethal concentration (as total metal concentration). During second year, the growth responses and tissue specific metals bio-accumulation in fish during chronic sub-lethal (1/3 of LC50 or LD50) water-borne and dietary exposure of individual metals and their mixture were studied for 12 weeks with three replications for each growth trail. The control fish were grown under metal free environment for comparison. The growth performance of each species of fish were monitored in terms of increase in wet weights, fork and total lengths, condition factor, feed intake and feed conversion ratios on weekly basis for 12 weeks under the above mentioned sub-lethal water-borne and dietary individual metals and metal mixture concentrations. Water quality characteristics viz. temperature, pH, hardness, dissolved oxygen, total ammonia, sodium, potassium and carbon-dioxide of the experimental aquaria were determined on daily basis. The fish body organs viz. kidney, liver, skin, muscle, fins, gills and bones were analyzed before and after 12-week growth trials, separately, exposed to water-borne and dietary sub-lethal metal concentrations. During third year, the growth responses and tissue specific metals bio-accumulation in fish under mixed (dual) toxicity of both water-borne and dietary metals were evaluated.

i)  Acute Toxicity of metals to the Fish

The sensitivity levels of three fish species, towards toxicity of water-borne copper, cadmium, zinc, nickel, cobalt and their mixture, were significantly different. The 120-day old fish were significantly least sensitive while 60-day fish showed significantly more sensitivity towards metal’s toxicity. All the three fish species were significantly least sensitive to cadmium. Among the three fish species, Catla catla showed significantly highest sensitivity towards zinc toxicity, followed by that of copper, metal mixture, nickel, cobalt and cadmium while Labeo rohita exhibited significantly least 96-hr LC50 for metal mixture, followed by that of copper, nickel, zinc, cobalt and cadmium. Cirrhina mrigala were significantly more sensitive to copper, followed by that of metal mixture, nickel, zinc, cobalt and cadmium. The sensitivity of fish to the dietary metals (both individual metals and metal mixture) decreased significantly with age. All 120-day three fish species showed significantly least sensitivity to dietary metals than that of 90- and 60-day fish. All the three fish species exhibited significantly highest sensitivity towards dietary copper. Both Catla catla and Labeo rohita exhibited significantly highest tolerance for dietary cobalt. However, Cirrhina mrigala were least sensitive to nickel. All the three fish species were significantly more sensitive to water-borne than dietary individual metals and their mixture. The relationships between water-borne (LC­50) and dietary (LD50) individual metals and metal mixture, for their impact on the tolerance limits of fish, were positively significant, illustrating strong relationships between them. However, acute toxicity of metals at higher concentrations caused significant excretion of ammonia by the fish resulting taxing conditions to enhance consumption of oxygen by the fish.

ii)   Growth responses and accumulation of metals in fish during water-borne and dietary
      exposures

During first hour of the growth period, both water- and diet-borne metal exposures exerted pronounced effects on the behavior of all the three fish species, indicating restiveness as compared with the control fish. All the fish grown under sub-lethal toxicity of metals/metal mixture showed hyperactivity and reduced exploratory behavior. However, these behavioral responses were significantly more pronounced in the fish exposed to water-borne metals/mixture than those exposed to dietary ones. The exposure of individual metals (copper, cadmium, zinc, nickel, cobalt) and metal mixture to the fish caused significant impact on their wet weight increments. However, the fish grown under metal free water (control) exhibited significantly better growth than those grown under either water-borne or dietary metal mixtures. Among the individual metals, cobalt and nickel exposures to the fish caused significantly lower weight gains. The metal mixtures, either water-borne or dietary intakes, caused significantly minimum growth in all the three fish species. The effects of metal mixtures, for their toxicity to the fish, differed significantly from the effects of a single metal. Even low concentration of metal mixture caused harmful impacts on fish growth, causing a variety of disturbances in their health and wellbeing as evident from the fish condition factor. Among the three fish species, Labeo rohita showed significantly higher sensitivity, in terms of weight escalations, towards metals toxicity, followed by Catla catla and Cirrhina mrigala with statistically significant differences. Water-borne metal exposures caused significantly lower weight gains in all the three fish species than those exposed to diet-borne metals. Weight increments of fish showed significantly positive relationship with feed intake under both water-borne and dietary metal exposures. The toxicity of different metals did not only influence the fish appetite but acclimation and feed conversion ratio that ultimately affected the fish growth significantly. Both water-borne and dietary intakes of metals have significantly affected the fish condition factor.

During growth trials, all the three fish species exhibited significantly variable responses towards accumulation of metals in their body organs and tissues. The exposure of water-borne individual metals caused significantly higher accumulation of all metals in three fish species than those exposed to dietary individual metals. The accumulation of metals was significantly higher in fish liver, followed by that in kidney. However, fish bones accumulated significantly lesser quantities of all the metals. All the three fish species showed significantly lesser tendency for the accumulation of cobalt in their body organs. Metal mixture exposure to all the three fish species resulted into higher concentrations of all metals in their liver while the same remained significantly lowest in fish bones.

iii)   Growth responses and accumulation of metals in fish during dual (mix) exposure of
        water-borne and dietary metals
The dual exposure of water-borne + dietary individual metals and their mixture elucidate significant effects on fish growth parameters. However, the effects of metal mixture differ in their toxicity from the effects of a single metal to all the three fish species under investigation. The toxicity of heavy metal mixture to the fish was dependent upon its concentration, specific composition and duration of exposure.
The present investigation elucidate significant effects of dual exposure of water-borne + dietary metals on the biological responses of fish which is a new bench mark regarding toxic responses of major carps to either individual metals or their mixture.

The exposure of fish to either individual metals viz. copper, cadmium, zinc, nickel, cobalt or their mixture resulted in significantly lower wet weight gains compared to the control. As far as overall responses of three fish species towards toxic responses of metals are concerned, metal mixture exposure exerted significant impact on fish, as evident from their low weight gains, followed by that of cadmium, copper, zinc, nickel and cobalt exposures. Among the treatments, copper stress resulted in significantly lower feed intakes in all the three fish species. The exposure of metal mixture to both Catla catla and Cirrhina mrigala, and cadmium to Labeo rohita caused significantly lesser feed conversion ratios. Regarding overall performance of fish in terms of FCR, both cadmium and metal mixture stresses to the fish caused significantly low FCR. However, cumulative FCR values of all the three fish species differed non-significantly. Among the three fish species, Catla catla were significantly more sensitive to metallic toxicity of dual nature (water-borne + dietary) while 30-day fish were significantly more sensitive to chronic effects of individual metals or their mixture.

Among the three fish species, Cirrhina mrigala performed significantly better in terms of increase in wet weights, fork and total lengths, specific growth rate (SGR) and feed intakes. However, FCR and fish condition factor did not show significant correlation with feed intake. However, SGR of fish had positively significant dependence on feed intake. Total ammonia had significantly direct relationship with fish weight escalations under Cu, Cd, Zn, Co and metal mixture exposures. The increase in carbon-dioxide contents of the test mediums has resulted in significant escalation of total ammonia contents in copper, nickel and control regimes. This shows higher oxygen consumption and ammonia excretion by the fish under chronic dual exposure of water-borne + dietary individual metals or their mixture.

Dual exposure of metals to the fish caused significantly higher accumulation of metals in liver, followed by that in kidney, gills, muscle, fins, skin and bones. Fish showed significantly higher tendency for the accumulation of both zinc and nickel. Among the three fish species, Catla catla exhibited significantly higher tendency to accumulate metals in its body, followed by Labeo rohita and Cirrhina mrigala. Fish liver and kidney appeared the prime sites of metal amassing while their loads in fish muscle were significantly lower than liver and kidney. Among the three age groups, 60-day fish exhibited significantly higher tendency towards accumulation of metals. Catla catla, Labeo rohita and Cirrhina mrigala exhibited significantly higher tendencies for the accumulation of nickel, cadmium and zinc, respectively. Catla catla accumulated less cadmium while that of both Labeo rohita and Cirrhina mrigala had the least concentrations of cobalt in their bodies after chronic exposure. The kidney and liver of all the three fish species responded significantly for the accumulation of all metals. Therefore, tissue-specific accumulation of metals can be proposed as a key indicator of chronic dual exposure of water-borne + dietary metals indicating the kinetics of metal accumulation in fish tissues during these investigations. Fish growth has been found a sensitive and reliable endpoint relating to simultaneous chronic exposures of water-borne and dietary individual metals and their mixture to three fish species viz. Catla catla, Labeo rohita and Cirrhina mrigala. Among the three fish species, Labeo rohita exhibited significantly higher tendency than that of Catla catla and Cirrhina mrigala to amass metals in their bodies. However, the difference between Catla catla and Cirrhina mrigala was statistically non-significant.