Experiments were conducted to determine the effects of excess dietary cobalt (Co) on its bioaccumulation, on digestive enzyme activities, and on the growth of freshwater Cyprinus carpio. Four isonitrogenous diets (average crude protein: 35 %) were formulated to prepare a control diet (T1) with no Co, and three Co-supplemented diets with 1.0 (T2), 1.5 (T3), and 2.0 (T4) % Co. The results showed that C. capio fed with T3 diet showed maximum apparent protein digestibility, feed conversion, protein utilization, and growth. Protease and lipase activities were maximum in the fish group under T3 diet. Accumulation of Co in different soft and hard tissues of the fish did not show any correlation with the level of Co in the diet. The concentration of Co in the water increased with its dietary level, being at an alarming level at 2.0 % dietary Co. It is concluded that an additional supply of dietary Co up to a level of 1.5 % is a viable option to augment growth of C. carpio, but at higher levels of Co it may be detrimental to the fish and the aquatic ecosystem.
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Keywords: cobalt; Cyprinus carpio; bioaccumulation; growth; mineral
The pollution of aquatic ecosystems by heavy metals is an important environmental problem (Rayms-Keller et al. 1998), as heavy metals constitute some of the most hazardous substances that can bioaccumulate (Tarifeno-Silva et al. 1982). Bioaccumulation is a process in which a chemical pollutant enters into the body of an organism and is not excreted, but rather collected in the organism's tissues (Zweig et al. 1999). Although some of these metals (e.g., Cu, Zn, Co) are essential trace elements for living organisms, they become toxic at higher concentrations. Therefore, fish become dependent on the diet for their supply. As a result, freshwater fish need adequate supply of minerals through diet (Hasan, 2000). Some of these heavy metals were used as micronutrient such as cobalt (Co).
Co is of relatively low abundance in the Earth's crust and in natural waters, from which it is precipitated as the highly insoluble Cobalt sulphite CoS. Although the average level of Co in soils is 8 ppm, there are soils with as little as 0.1ppm and others with as much as 70 ppm. In the marine environment Co is needed by blue-green algae (cyanobacteria) and other nitrogen fixing organisms (Watanabe et al.1997).
Cobalt (Co) is one of the 13 minerals that have been demonstrated to be essential components in the diet of fish (Davis and Gatlin, 1991). Co is an essential metal as an integral component of cyanocobalamin (vitamin B12) constituting nearly 4.5% of its molecular weight which is indispensable for animal growth. Most animals need Co for vitamin B12 synthesis by intestinal microflora. It also influences certain enzymes, binds to insulin (Cunningham et al.1955) and also reduces plasma glucose levels (Roginski and Mertz, 1977).It is a part of vitamin B12 and is required for nitrogen assimilation, erythrocyte maturation, and synthesis of hemoglobin (Hazell, 1985). Intestinal micro-organisms help to synthesize vitamin B12, but absence of Co in the diet lower intestinal synthesis and liver stores of vitamin B12 (Limsuwan and Lovell, 1981). Deficiency of vitamin B12 causes several abnormalities including poor appetite, poor growth, low hemoglobin, and anemia in fish (Stickney, 1994). Co also functions as a cofactor and activator for a number of enzymes.
Since freshwater fish are capable of absorbing minerals from the surrounding water in addition to the food ingested, the dietary requirement of a freshwater fish species for a particular element depends to a large extent on the concentration of the element in the medium (Hepher 1990). Co has been reported to be extremely scarce in freshwater bodies in different parts of the world (Baralkiewicz and Siepak, 1999; Karadede-Akin and Unlu, 2007). Because of this uncertainty, freshwater fish require dietary supplements of Co for optimum growth. Previous studies reported results on Co accumulation and their toxicity effect in aquatic organisms (Nolan et al. 1992). Co acute to chronic ratios determined for Daphnia were 111 and 1163 in soft water and hard water, respectively (Diamond et al. 1992) indicating large differences between acute and chronic effects thresholds. Co toxicity data also exist for several fish species goldfish (Carassius auratus), fathead minnow (Pimephales promelas), zebrafish (Brachydanio rerio), and banded gouramis (Colisa fasciatus) (Marra et al.1998). Baudin and Fritsch (1989) examined the related contribution of food and water in the accumulation of cobalt in fish. Carp fed Co-contaminated snails were found to accumulate Co only slightly. Marra et al. (1992) reported that a clear dose-response relationship between Co concentration and mortality, although rainbow trout did not start dying in concentrations that eventually produced 100 % mortality until after the first 2 days.
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Fish accumulated Co-cobalamine 21 times more rapidly from seawater than CoCl2 and retained ingested Co-cobalamine 20 times more efficiently (100 %) than ingested CoCl2 (5 %). Two thirds of the ingested Co-cobalamine was retained in the fish with a half-time of 8 d. The remaining one third of the organic form was retained with a half-time of 54 d, a value which was not significantly different from that of CoCl2 (47 d) (Nolan et al. 1992). On the other hand, very high doses of Co (0.1-5 g Co kg-1) were toxic to rainbow trout, resulting in haemorrhages in the digestive tract and alterations in white blood cells (Watanabe et al.1997). Co deprivation reduced the intestinal synthesis of vitamin B12 in catfish (Limsuwan and Lovell, 1981). Nevertheless, there is little information regarding the balance of Co in the fish (Hasan 2000). Although animal and plant feed stuff used in artificial feed formulations generally provide adequate quantities of minerals (Gatlin and Wilson, 1986) some species require additional supplies (Lorentzen and Maage, 1999). There is evidence that fish reared with Co-supplemented diet (CSD) show significant increase in growth over fish fed with control diet without supplement (Anadu et al. 1990; Mahmoud 2009). Limsuwan and Lovell (1981) found enhanced growth in channel catfish fed with 1.4 mg kg-1 Co-supplemented feed. Therefore, this study is aimed to assess to screen the supplementation of Co to ensure that the metal is not accumulated to unnecessarily high levels eliciting toxicological effects.
2.1. Experimental diets
An experimental diet were prepared with raw ingredients such as rice bran, wheat flour, mustard oil cake, fishmeal, vitamin premix, and mineral premix without Co (2%) and was formulated to contain approximately 35% crude protein. Cobalt (II) chloride hexahydrate was added in required quantity to the prepared diet to make four experimental diets with four different levels of dietary Co: 0.0% (T1), 1.0 % (T2), 1.5 % (T3), and 2.0 % (T4). To test protein digestibility of the diets, 1.0% chromic oxide (Cr2O3) were added to each diet separately. All diets were prepared in pelleted form using 0.5% carboxymethyl cellulose as a binder and the pellets were sun-dried for a few days before use in the trial. Samples of each diet were digested in strong nitric acid, perchloric acid, and sulfuric acid (Churnoff, 1975) and the level of Co in the digested sample was determined in atomic absorption spectrophotometer (AAS).
2.2. Experimental design
Two different feeding trials were made with the fingerlings of C. carpio: a digestibility trial and a growth trial. Fingerlings of the fish were collected from local hatchery and their initial morphometric characteristics were recorded. The fingerlings were stocked in 50-L glass aquaria containing deep tube-well water stored in an overhead tank. The fish was acclimatized to this condition for 1 week before using in any trial.
2.3. Digestibility trial
The digestibility trial was conducted in 15-L glass aquaria in the laboratory. Altogether 12 aquaria were arranged according to randomized block design with three replicates for each of the four dietary conditions. Each aquarium was stocked with four fishes. The fish was fed with a ration at 6% of their body weight. The ration was provided at 0800 h and the fish was allowed to eat for 6 h. Left over diets was collected after 6 h of feeding, oven-dried, and weighed. The leaching rate was estimated by placing weighed diets in aquaria without fish for 6 h and then recollecting, drying, and re-weighing the diets. To minimize nutrient leaching, only fresh and intact feces was collected and dried to a constant weight at 60°C in an oven and weighed before preserving at -20°C. The trial was continued for 1 week. The preserved dried diet and feces was brought to room temperature, weighed, and ground in a mortar to make a pooled sample for each replicate. Chromium was extracted from the pooled sample of feces and of the diets using sulfuric acid, perchloric acid, and molybdenum sulfate (Bolin et al. 1952) and chromium (Cr) content in the sample was determined in Flame AAS. Apparent protein digestibility (APD) of the diet was calculated from the proportion of Cr and protein in the diet and feces following the methods described by Ellestad et al. (2002) and Mondal et al. (2008).
2.4. Growth trials
The growth trials were made in 400 L outdoor cement tanks. Each tank was stocked with 40 acclimatized fingerlings. Altogether 12 tanks were arranged according to randomized block design so that fingerlings could be reared in three replicates for each of the four dietary conditions. The fish was fed twice daily at an interval of 8 h at a fixed ration of 6% of the body weight. Samples of fish were weighed after every 10 days and the quantity of the feed required for each tank be readjusted. Mortality of the fishes was checked daily and the dead fishes, if any, were removed immediately to avoid decomposition. All fish from each outdoor tank was sampled at the end of 8 weeks trial; the length and weight of the fish was recorded and five sampled fish from each tank was subjected to biochemical analyses to determine moisture, crude protein, lipid, and ash content (g kg-1 wet weight basis) of the fish. Percent increase in weight (PIW), specific growth rate (SGR% per day), feed conversion ratio (FCR), protein efficiency ratio (PER), and Net protein utilization (NPU%) was calculated using standard methods (Steffens, 1989).
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Samples of water were collected from each tank on day 1 and every week thereafter for the determination of water quality parameters. The parameters determined include temperature, pH, dissolved oxygen (DO), total ammonia nitrogen (TAN) and nitrates (NO3-N). Standard methods of APHA (1995) were followed for these determinations.
2.5. Digestive enzyme assay
Samples of fish for digestive enzyme analyses were collected at the start (initial) and at the end of the trials. Fish samples used for digestive enzyme analyses were kept alive in glass aquaria containing clean water and was starved for 24 h. After 24 h of starving, the fish was sampled. Intestine of the fish was collected, placed on ice, washed several times in sterile, chilled saline, soaked, and weighed. The tissue was then homogenized and sonified to make intestinal homogenates. Triplicate samples per treatment were maintained. The intestinal homogenates was then used to extract the digestive enzymes - agr-amylase, protease, and lipase, respectively, by the methods of Bernfeld (1955), Marks and Lajtha (1963) and Josep and Kurup (1999). Protein content of the intestinal tissue was determined by the method of Lowry et al. (1951).
2.5. Bioaccumulation of Co
Samples of water and fish were collected before the start of the experiment (initial) and at the end of the experiment to determine level of Co in water and in different tissues of the fish. The water samples were digested by strong nitric acid (APHA 1995). The sampled fish was soaked in blotting paper and dissected with a clean pair of acid soaked scissors to collect liver, kidney, gill, muscle, and bone (vertebral column) tissues. The tissues was wrapped in polyethylene paper and preserved at -20°C before digestion for determination of Co. During digestion, the tissues were brought to room temperature, thawed, and digested in strong nitric acid, perchloric acid, and sulfuric acid, following the method of Churnoff (1975) and Paacuteez-Osuna and Tron-Mayen (1995).
2.6. Analytical procedures
Proximate composition analyses of the experimental diets as well of carcass were performed following the AOAC (1992).
2.7. Statistical analysis
Treatment effects were compared by the least significant difference method using MstatC software of Michigan State University, MI, USA. Significance of difference has been presented in the form of probability (P) values. Treatments were compared, (LSD) to determine significant variation between the dietary levels (Gomez and Gomez, 1984).
3. Results and Discussion
Actual level of Co detected in the diets were not detectable (ND) under T1 (0.0%), T2 (1.0%), T3 (1.5%), and T4 (2.0%) diets. Apparent protein digestibility (APD) of the control (72.4 ± 0.05), T2 (78.5 ± 2.4), and T4 (90.6 ± 1.8) diets were similar, while APD significantly increased in T3 (P ≤ 0.1%) diet (90.6 ± 1.8) as compared to control diet (Table 1). Fingerlings of C. carpio reared with diets containing additional Co (T2-T4) showed significantly higher growth than those reared with control diet (Table 1). Results of ANOVA showed significant variation of the growth parameters between the treatments (diet groups). Comparing mean values of these parameters between the diet groups by LSD test revealed that growth was significantly higher (higher PIW, SGR, PER, and lower FCR) in fish fed under T3 as compared to control T1, T2, and T4. Food conversion Ratio (FCR) between T2 and T3 was significantly different (P ≤ 0.05%). There was non -significant difference in these growth parameters between T2 and T4 treatments. Although Net protein digestibility (NPU), like other growth parameters, was maximum in T3, it significantly varied between all pair of treatments, T2-T4 treatments showing significantly higher values than the control (T1). The results of this study indicate that the dietary supplement of Co up to a level of 1.5% is a viable option to augment the growth of C. carpio. The maximum digestibility of protein (APD) of the diet at inclusion level of 1.5% Co (T3) was correlated with the highest growth of the fish fed this diet. Inadequate literature sources are available to quantify dietary requirement of cobalt for optimum growth of fish. While a few species like rainbow trout requires meager amount of Co (0.05 mg Co · kg-1 diet; Hasan 2000) effects of the mineral on growth of fish are not well established. Diets used for common carp in the present investigation contained Co in much higher level than those required by rainbow trout. This excess dietary cobalt increased the digestibility of the diets and growth of common carp. The findings of the present study were seems in line with those as reported by Mukherjee and Kaviraj (2009). Sukhoverkhov (1967) found increased growth of common carp fingerlings and of 2-year-old fish fed diet containing even much lower level of dietary Co (0.03%). Hertz et al. (1989) also found improved growth and Protein Efficiency Ratio (PER) of C. carpio fed Co chloride as part of a low protein diet. There are also reports of growth promoting effect of dietary Co on other species of fish (Mahmoud, 2009). The growth rate of O. niloticus was increased when the fish were fed on diets containing small doses (0.01 mg/day/fish) of cobalt salt (Mahmoud, 2009). The results indicate that Co act as growth promoter for common carp. Anadu et al. (1990) found higher growth in Tilapia zilli fed diets supplemented by Co than the control fish. Adhikari and Ayyappan (2002) observed that Indian major carp Labeo rohita exhibited significantly higher growth when Co was used as a micronutrient fertilizer. The growth rate of C. carpio in this study was improved by adding CoCl upto 1.5 % to supplementary diets under T3 and could be considered as viable option. The results indicate that Co act as growth promoter for common carp. Growth promoting effects of Co has also been demonstrated for and rainbow trout (Hossein et al. 2008).
The proximate composition of the carcass at the end of the trial showed a significant increase in crude protein level from the initial values in all the dietary groups (T1-T4; Table 2). The crude protein increase was significantly higher (P < 0.05) in all the Cobalt supplementary diets (CSDs) (T2-T4) as compared to the control diet (T1). Maximum increase was observed in (T3) followed by T4 and T2. There was significant difference between T2 and T4. Crude lipid level of the carcass significantly increased only in T1, T3, and T4 treatments. There were non-significant differences between T1 and T4 for crude lipid content. There was no significant difference in the ash content of the body between initial values and the T2 and T1, T3. Ash content significantly increased from the initial value in T3. Yildiz (2008) also observed significantly higher level of Co in fillet of fish cultured on diets containing Co as compared with those captured from the wild. In the present study dietary inclusion of Co at 1.0% (T3) level, however, significantly increased Co in all tissues.
The mean values of the digestive enzyme activities in the intestine of C. carpio are given in Table 3. All enzyme activities significantly increased from the initial concentration determined before beginning the experiment. Maximum agr-amylase activity (12.8 mg maltose liberated (mg protein-1) (h-1)) was found in fish fed T2 diet followed by T3, T4, and T1 diet (Table 3). Maximum protease activity (44.6 µg histidine liberated (mg protein-1) (h-1)) was found in T3 followed by T4, T2, and T1 diets. Highest lipase activity (11.4 mg protein min-1) was also found in T3 followed by T4, T2, and T1 treatments. Existing literature have scanty information responding to dietary Co in Cyprinus carpio. Mahmoud (2009) reported growth-promoting effects of Co in channel catfish. The metabolic changes resulting from the effects of diet are influenced by the digestive enzyme activities of the fish (Keshavanath et al. 2003). The gut enzyme profile is thus functionally linked to fish performances (Nwanna, 2007). Giri et al. (2000) detected agr-amylase activity in the catfish Clarias batrachus indicating that catfish are also able to digest starch efficiently. In this study Cyrinus carpio showed higher amylase activities in all the treatments than the control diet. Dietary mineral supplements have also been demonstrated to increase amylase activities in hybrid tilapia Oreochromis niloticus times Oreochromis aureus (Li et al. 2007). However, protein and lipids are pivotal in the diet of most carnivorous fish (Chou et al. 2001) and thus proteolytic and lipolytic activities in the gut are essential clue to the performance of the fish. Cyprinus carpio fed Zn supplements, significantly increased intestinal trypsin (Brafield and Koodie, 1994), whereas, supplementation of Cu, Fe, or Zn had no effect in hybrid tilapia (Li et al. 2007). In this study, protease activities significantly increased in C. carpio till the diet contained 1.0% dietary Co. Further increase in the level of dietary Co (e.g., 1.5%) decreased the protease activity. Very high doses of Co (0.1-5 g Co kg-1) were toxic to rainbow trout, resulting in hemorrhages in the digestive tract and alterations in white blood cells (Watanabe et al. 1997). Lipase has been identified in catfish intestine (Lundstedt et al. 2002). The results of this study indicated that lipase activities of C. carpio increased in all the Co-supplemented diets (T2-T4) as compared to control diet. Maximum lipase activity and best growth of C. carpio were however observed under T3 indicating that the fish are capable of digesting maximum lipid when the feed was supplemented by 1.0% Co. The occurrence of lipase activity is more important in fish as they feed on food rich in fat (Chakrabarti et al. 1995). It may be argued that fish utilize fat as an important nutritional source rather than carbohydrate and protein. Nevertheless, mineral supplement in diet influences lipase activity in the fish as observed for Co supplement in this study.
Co could not be detected in water before the start of the experiment (initial) and at the end of 8 weeks of trial in control group (T1). Concentration of Co detected in water after 8 weeks of trial in T2, T3, and T4 were, respectively, 0.2 ± 0.03, 0.8 ± 0.2, and 1.9 ± 0.4 mg L-1. Concentration of Co determined in liver, kidney, gill, muscle and bone before the start (initial) of the trial were, respectively, 5.22 ± 0.9, 7.23 ± 0.88, 3.11 ± 0.18, 5.60 ± 0.12, and 9.15 ± 1.23 µg g-1 (Table 4). None of the tissues showed any correlation between the concentration of Co detected in tissues at the end of trial and level Co in the diet. Single-factor ANOVA showed that concentration of Co in liver, kidney, gill, muscle and bone tissues vary significantly between the diet groups (P > 0.05). However, muscle (12.76 ± 1.87 µg g-1) and bone tissues (13.10 ± 1.66 µg g-1) showed a significant increase in Co accumulation in fish fed under T3, though concentrations of Co detected in these tissues in T4 as 19.51 ± 4.34 µg g-1 in muscle and 31.91 ± 5.64 µg g-1 in bone (Table 4). Results of this study did not indicate any pattern of Co accumulation form the diet in C. carpio. This is in contrast to C. carpio which showed the capability of maintaining equilibrium between uptake of Co from diet and its excretion, thereby, showing almost a constant concentration in most of the tissues up to dietary level of 1.5 % Co under T3 (Akan et al. 2009). The result is in line with the work of (Buckley et al. 1982) who indicated that in fish, the liver is the major storage organ for cobalt. Accumulation in the liver can be the result of detoxicating mechanisms and may originate from metal in the food (Karadede and Unlu, 2007; Shoham-Frider et al. 2002). Generally, exposure of fish to cobalt in water results in 50 % of the metal being associated with the skin and external organs (Nolan et al. 1992). Co is essential as an integral component of vitamin B12 which is indispensable for fish growth (Miyazaki et al., 2001). Co was reported to accumulate in many tissues such as liver, gonads, and digestive duct (Nolan et al. 1992). Different fish tissues have got different Co accumulation capacities. In this study total Co accumulation in the body tissues were analyzed. However, the liver is the preferred organ for metal accumulation as could be deduced from the present study. However, this study revealed that concentration of Co significantly increased in all tissues of C. carpio when dietary level of Co increased to 1.0%. In this study, C. carpio did not show any correlation between concentration of Co in tissues and level of Co in the diet. Initial concentration of Co in different tissues of C. carpio observed in this investigation was higher than those observed in C. carpio (Guner, 2010). We could not ascertain any reason of such high initial concentration of Co as well as significantly higher level of Co in muscle and bone in 1. 0 % CSD(T2). But high background concentration probably prevented excess accumulation of Co in gill, liver, and kidney from the diet. Concentration of Co detected in water in T4 diet group (2.0 % Co) is alarming because this level of Co in water may affect survival and growth of algae (Colemanand Rice 1971; Akan et al. 2009) and crustacean zooplankton (Das and Kaviraj, 1994).
Average weekly values of water quality parameters (temperature 3.5-38.5°C; pH 7.4-7.9; DO 5.6-6.4 mg L-1; total NH3-N 0.06-0.10 mg L-1; NO3-N 0.04-0.09 mg L-1) recorded during the trial were within the optimum ranges required for rearing C. carpio and did not vary between the dietary groups.
Fish absorb metals through ingestion of water or contaminated food. Co has been shown to undergo bioaccumulation in the tissue of fish under this experiment. On consumption of fish and other aquatic organisms these metals become transferred to man. However, it is not yet known whether the fishes in the freshwater bodies have been severely affected by heavy metals based on the results obtained from this study. Although the results do not explicitly indicate a manifestation of toxic effects, the possibility that deleterious effects could manifest after a long period of consumption of fish caught in with trace metal contamination cannot be ruled out.
The authors gratefully acknowledge Research Centre, College of Sciences and Humanities Studies Al-Kharj University, Kingdom of Saudi Arabia, for providing funding to carry out the research work.